Abstract:

The invention provides reagents and methods for inhibiting or enhancing
interactions between proteins in hematopoietic cells and other cells
involved in the mediation of an immune response. Reagents and methods
provided are useful for treatment of a variety of diseases and conditions
mediated by immune system cells.

Claims:

1-48. (canceled)

49. A method of determining whether a test compound is an inhibitor or
agonist of binding between a PDZ protein and a PL protein, comprising:(a)
contacting a peptide comprising a PDZ domain (PDZ) with a plurality of PL
peptides (PL) in the presence and in the absence of a test compound,(b)
detecting any interaction between the PDZ and at least one PL in the
presence and absence of the test compound,wherein(i) less interaction
between the PDZ and at least one PL in the presence of the test compound
than in the absence of the compound indicates that the test compound is
an inhibitor of such an interaction, or(ii) more interaction between the
PDZ and at least one PL in the presence of the test compound than in the
absence of the compound indicates that the test compound is an agonist of
such an interaction.

50. The method of claim 49, wherein the PDZ is contacted with the
plurality of PLs simultaneously.

51. The method of claim 50, wherein the PLs are organized in an array.

52. The method of claim 51, wherein the different PLs are situated in
separate areas of the array.

53. The method of claim 51, wherein the different PLs are each situated in
a different well of a multi-well plate.

54. The method of claim 51, wherein the PL peptides are arrayed within a
common area of a surface such that they can be simultaneously exposed to
the same solution.

55. The method of claim 51, wherein the method comprises the use of
multiple arrays of PLs in tandem.

56. The method of claim 49, wherein at least one of the plurality of PLs
is immobilized.

57. The method of claim 56, wherein the at least one of the plurality of
PLs is bound to the surface of an assay plate.

58. The method of claim 57, wherein the assay plate is a multi-well plate.

59. The method of claim 57, wherein the assay plate is a high density
array.

60. The method of claim 49, wherein the test agent comprises a peptide
having at its carboxy-terminus at least two amino acids that are the same
as the carboxy-terminal two residues of a PL protein.

61. The method of claim 60, wherein the test agent comprises a motif
X-S-X-A, X-A-D/E-V, X-V/I/L-X*-V, or X-S/T-X-F, (where X is any amino
acid and X* is any non-aromatic amino acid).

62. The method of claim 49, wherein at least one of the plurality of PLs
is bound to a substrate in the form of a distinguishable bead.

63. The method of claim 49, further comprising determining the effect on
the test agent upon a plurality of PDZ-PL pairs.

64. The method of claim 63, wherein the determining is performed
simultaneously on the plurality of PDZ-PL pairs.

65. The method of claim 64, further comprising selecting a test agent that
modulates the binding of a first PDZ-PL pair of interest, but does not
inhibit, or inhibits to a lesser extent, the binding of a second
different PDZ-PL pair of interest.

66. The method of claim 65, wherein the first PDZ-PL pair comprises the
same PDZ as the second PDZ-PL pair, but comprises a different PL.

67. The method of claim 65, wherein the first PDZ-PL pair comprises a
different PDZ and a different PL from the second PDZ-PL pair.

68. The method of claim 49, wherein the method involves determining
whether a test compound is an inhibitor.

Description:

REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. application Ser. No.
09/688,017, which is (1) a continuation-in-part of U.S. application Ser.
Nos. 09/570,118, 09/570,364, and 09/569,525 (all filed May 12, 2000),
each of which claims the benefit of U.S. Provisional Application Nos.
60/196,460, 60/196,528, 60/196,527, and 60/196,267 (all filed Apr. 11,
2000), and (2) a continuation-in-part of U.S. application Ser. No.
09/547,276, filed Apr. 11, 2000, which claims the benefit of U.S.
Provisional Application Nos. 60/182,296 (filed Feb. 14, 2000); 60/176,195
(filed Jan. 14, 2000); 60/170,453 (filed Dec. 13, 1999); 60/162,498
(filed Oct. 29, 1999); 60/160,860 (filed Oct. 21, 1999); and 60/134,118;
60/134,117; and 60/134,114 (all filed May 14, 1999); the disclosures of
each of which are incorporated herein in their entirety.

1. FIELD OF THE INVENTION

[0002]The present invention relates to peptides and peptide analogues, and
methods for using such compositions to regulate activities of cells of
the hematopoietic system. In one aspect, the invention provides methods
of modulating metabolism (e.g., activation) of hematopoietic cells (e.g.,
T cells and B cells) by antagonizing an interaction between a PDZ domain
containing protein and a protein that binds a PDZ domain. In one aspect,
it relates to fusion peptides containing an amino acid sequence
corresponding to the carboxyl terminus of a surface receptor expressed by
a hematopoietic cell and a transmembrane transporter sequence; such
fusion peptides are useful in regulating hematopoietic cells by
inhibiting cell activation.

[0004]PDZ domains have been shown to bind the carboxyl termini of
transmembrane proteins in neuronal cells. Songyang et al. reported that
proteins capable of binding PDZ domains contain a carboxyl terminal motif
sequence of E-S/T-X-V/I (Songyang et al., 1997, Science 275:73). X-ray
crystallography studies have revealed the contact points between the
motif sequence and PDZ domains (Doyle et al., 1996, Cell 88:1067-1076).
While the interaction between PDZ domains and ion channels in neurons
have been studied extensively, such interactions have had limited studies
in other biological systems, especially the hematopoietic system.

[0005]The hematopoietic system is composed of different cell types that
perform distinct functions. Many of its diverse function requires
coordinated movement of cell surface receptors including ion channels,
adhesion surface molecules to coordinate cell-cell interaction, and
cytokine receptors. Despite their diverse functional activities, all
hematopoietic cells are believed to develop from a multipotent bone
marrow hematopoietic stem cell. Such stem cell has been shown to express
a surface marker termed CD34. During differentiation, the stem cell gives
rise to progenitor cells in each of several specific hematopoietic cell
lineages. The progenitor cells then undergo a series of morphological and
functional changes to produce mature functionally committed hematopoietic
cells.

[0006]Among the functions performed by hematopoietic cells, certain cell
types are involved exclusively in immunity. For example, lymphocytes,
which include T cells, B cells and natural killer (NK) cells, are
effectors in immune responses. Monocytes and granulocytes (i.e.,
neutrophils, basophils and eosinophils) play a role in non-specific forms
of defense. Lymphocytes, monocytes and granulocytes are collectively
referred to as white blood cells or leukocytes. On the other hand, other
hematopoietic cells perform functions that are unrelated to the immune
system. For example, erythrocytes are involved in gas transport, and
cells of the thrombocytic series are involved in blood clotting.

[0007]T cells and B cells recognize antigens and generate an immune
response. T cells recognize antigens by heterodimeric surface receptors
termed the T cell receptor (TCR). The TCR is associated with a series of
polypeptides collectively referred to as CD3 complex. B cells recognize
antigens by surface immunoglobulins (Ig), which are also secretory
molecules. In addition, a large number of co-stimulatory surface
receptors have been identified in T cells and B cells, which augment
cellular activation during antigen-induced activation.

[0009]Specific antibodies have been generated against all of the
aforementioned T cell surface antigens. Other molecules that bind to the
aforementioned T cell surface receptors include antigen-binding antibody
derivatives such as variable domains, peptides, superantigens, and their
natural ligands such as CD58 (LFA-3) for CD2, HIV gp120 for CD4, CD27L
for CD27, CD80 or CD86 for CD28 or CD152, ICAM1, ICAM2 and ICAM3 for
CD11a/CD18, 4-1BBL for CDw137.

[0010]Activation molecules expressed by B cells, include but are not
limited to, surface Ig, CD18, CD19, CD20, CD21, CD22, CD23, CD40, CD45,
CD80, CD86 and ICAM1. Similarly, natural ligands of these molecules and
antibodies directed to them as well as antibody derivatives may be used
to deliver an activation signal to B cells.

[0011]However, prior to the present invention, it was not known that
signal transduction following stimulation of any leukocyte receptor was
mediated by receptor interactions with PDZ domain-containing proteins.
Therefore, it was not even contemplated in the art that an interference
of leukocyte surface receptor/PDZ domain interactions could regulate
leukocyte activation.

3. SUMMARY OF THE INVENTION

[0012]In one aspect, the invention provides a method of modulating a
biological function of a cell, e.g., an endothelial cell or hematopoietic
cell (such as a leukocyte, e.g., T cell or B cell), by introducing into
the cell an antagonist that inhibits binding of a PDZ protein and a PL
protein in the cell, or a agonist that enhances binding of a PDZ protein
and a PL protein in the cell. In various embodiments the PL protein is an
adhesion protein, an adaptor protein, or an intracellular protein. In
embodiments it is CD6, CD49E, CD49F, CD138, CLASP-1, CLASP-4, VCAM1,
CLASP-2, CD95, DNAM-1, CD83, CD44, CD4, CD97, CD3n, DOCK2, CD34, FceRIb,
or FasLigand. In an embodiment the PL protein is characterized by a
carboxy-terminal amino acid motif that is X-S-X-A, X-A-D/E-V,
X-V/I/L-X*-V, or X-S/T-X-F (where X is any amino acid and X* is any
non-aromatic amino acid). In embodiments, the PL protein is expressed by
T lymphocytes or B lymphocytes. In some embodiments of this method, the
PDZ protein is CASK, MPP1, DLG1, PSD95, NeDLG, SYNla, TAX43, LDP, LIM,
LIMK, AF6, PTN-4, prIL16, 41.8, RGS12, DVL1, TAX 40, TIAM1, MINT1, K303,
TAX2, or KIAA561.

[0013]In some embodiments, the cell is a leukocyte and the biological
function is cell activation, cell proliferation, maintenance of cell
structure, cell metabolic activity, or cytokine production. In some
embodiments, the method further includes detecting a change in leukocyte
activation.

[0014]In preferred embodiments, the antagonist is an agent that inhibits
the binding of a PL peptide to a PDZ domain polypeptide in an "A" assay,
in a "G" assay, or in both an A assay and a G assay. The antagonist can
be a polypeptide, such as a polypeptide having at the carboxyterminus at
least two residues that are the same as the carboxy-terminal two residues
of a PL protein, such as a PL protein is expressed in a hematopoietic or
endothelial cell that is an adhesion protein, an adaptor protein, or an
intracellular protein. In an embodiment, at least the carboxy-terminal
four residues of the polypeptide are the same as the carboxy-terminal
four residues of the PL protein. In an embodiment, the PL protein has a
carboxy-terminal amino acid motif selected from X-S-X-A, X-A-D/E-V,
X-V/I/L-X*-V, or X-S/T-X-F, where X is any amino acid and X* is any
non-aromatic amino acid. In embodiment, the PL protein is CD6, CD49E,
CD49F, CD138, CLASP-1, CLASP-4, VCAM1, CLASP-2, CD95, DNAM-1, CD83, CD44,
CD97, CD3n, DOCK2, CD34, FceRIb, or FasLigand.

[0015]In a related aspect, the antagonist is a peptide mimetic of a PL
inhibitor sequence peptide. In another related aspect the antagonist is a
fusion polypeptide having a PL sequence and transmembrane transporter
amino acid sequence (such as HIV tat, Drosophila antenapedia, herpes
simplex virus VP22 or anti-DNA CDR 2 and 3).

[0016]In another aspect, the invention provides a method of determining
whether a test compound is an inhibitor of binding between a PDZ protein
and a PL protein by contacting a PDZ domain polypeptide having a sequence
from the PDZ protein, and a PL peptide under conditions in which they
form a complex, in the presence and in the absence of a test compound,
and detecting the formation of the complex in the presence and absence of
the test compound, where less complex formation in the presence of the
test compound than in the absence of the compound indicates that the test
compound is an inhibitor of a PDZ protein-PL protein binding. The PL
peptide has a sequence that includes the a C-terminal sequence of a PL
protein, such as CD6, CD49E, CD49F, CD138, CLASP-1, CLASP-4, VCAM1,
CLASP-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, or
FasLigand. In some embodiments, the PDZ domain polypeptide is a fusion
polypeptide.

[0017]In a related aspect, the invention provides a method of determining
whether a test compound is an agonist of binding between a PDZ protein
and a PL protein by contacting a PDZ domain polypeptide, and a PL peptide
under conditions in which they form a complex, in the presence and in the
absence of a test compound, and detecting the formation of the complex in
the presence and absence of the test compound, where more complex
formation in the presence of the test compound than in the absence of the
compound indicates that the test compound is an agonist of a PDZ
protein-PL protein binding.

[0018]The invention further provides an inhibitor of binding of a PDZ
protein and a PL protein. In an embodiment, the inhibitor is
characterized in that it reduces binding of a peptide selected from the
group consisting of a PL peptide selected from the group consisting of
CD6, CD49E, CD49F, CD138, CLASP-1, CLASP-4, VCAM1, CLASP-2, CD95, DNAM-1,
CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand and a PDZ
domain polypeptide. In various embodiments, the inhibitor is a peptide
comprising a sequence that is from 3 to about 20 residues of a C-terminal
sequence of a PL protein selected from CD6, CD49E, CD49F, CD138, CLASP-1,
CLASP-4, VCAM1, CLASP-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2,
CD34, FceRIb, and FasLigand; a peptide having a motif X-S-X-A, X-A-D/E-V,
X-V/I/L-X*-V, or X-S/T-X-F, (where X is any amino acid and X* is any
non-aromatic amino acid); a peptide mimetic; or a small organic molecule.
The invention also provides a pharmaceutical composition containing the
inhibitor.

[0019]The invention also provides a method for treating a disease
characterized by leukocyte activation by administering a therapeutically
effective amount of an inhibitor of a PL-PDZ interaction. In embodiments,
the disease is characterized by an inflammatory or humoral immune
response or is an autoimmune disease. The invention further provides a
method of reducing inflammation in a subject, by administering an agent
that inhibits binding of a PDZ protein and a PL protein, where the PL
protein is an adhesion protein, an adaptor protein, or an intracellular
protein.

[0020]The invention also provides use of an inhibitor of the binding of a
PDZ protein and a PL protein to inhibit leukocyte activation or to treat
a disease mediated by hematopoietic cells, such as a disease is
characterized by an inflammatory or humoral immune response. The
invention also provides use of an inhibitor of the binding of a PDZ
protein and a PL protein in the preparation of a medicament for treatment
of a disease mediated by hematopoietic cells.

[0021]The invention also provides a method of modulating a biological
function of a hematopoietic cell, comprising introducing into the cell an
antagonist that inhibits binding of a PDZ protein and a PL protein in the
cell as deduced from Table 2, for example, where the PL protein is DNAM-1
and the PDZ protein is MPP1, MPP2, DLG1, NeDLG, PSD95, LIM, AF6, 41.8 or
RGS12, the PL protein is LPAP and the PDZ protein is DLG1 or MINT1, or
the PL protein is DNAM-1 and the PDZ protein is PSD95 or MPP2.

[0022]The present invention also relates to peptides and peptide analogues
that bind PDZ domains in hematopoietic cells. In particular, it relates
to fusion peptides and peptide analogues containing a hematopoietic cell
surface receptor carboxyl terminal sequence and a transmembrane
transporter sequence which facilitates entry of the peptides into a
target cell. The invention also relates to methods of using such
compositions in inhibiting leukocyte activation as measured by cytokine
production, cell proliferation, apoptosis and cytotoxicity.

[0023]It is an object of the invention to administer a therapeutically
effective amount of the aforementioned fusion peptides or peptide
analogues as pharmaceutical compositions to a subject to inhibit
undesirable leukocyte-mediated events.

[0024]It is also an object of the invention to administer a
therapeutically effective amount of the aforementioned fusion peptides or
peptide analogues as pharmaceutical compositions to a subject to treat an
autoimmune disorder or to prevent transplantation rejection of a solid
organ transplant.

[0025]In one aspect, the invention provides a method of determining the
apparent affinity (Kd) of binding between a PDZ domain and a ligand, by
(a) immobilizing a polypeptide comprising the PDZ domain and at least one
non-PDZ domain on a surface; (b) contacting the immobilized polypeptide
with a plurality of different concentrations of the ligand; (c)
determining the amount of binding of the ligand to the immobilized
polypeptide at each of the concentrations of ligand; (d) calculating the
apparent affinity of the binding from the binding determined in (c). In
an embodiment, the polypeptide is immobilized bybinding the polypeptide
to an immobilized immunoglobulin that binds the non-PDZ domain. In an
embodiment, the polypeptide comprising the PDZ domain is a fusion
protein, for example a GST-PDZ domain fusion protein.

[0026]In one aspect, the invention provides a method of determining the Ki
of an inhibitor or suspected inhibitor of binding between a PDZ domain
and a ligand, by (a) immobilizing a polypeptide comprising the PDZ domain
and a non-PDZ domain on a surface; (b) contacting the immobilized
polypeptide with a plurality of different mixtures of the ligand and
inhibitor, wherein the different mixtures comprise a fixed amount of
ligand, at least a portion of which is detectably labeled, and different
concentrations of the inhibitor; (c) determining the amount of ligand
bound at the different concentrations of inhibitor; (d) calculating the
Ki of the inhibitor from the binding determined in (c). In an embodiment,
the polypeptide is immobilized by binding the polypeptide to an
immobilized immunoglobulin that binds the non-PDZ domain. In an
embodiment, the fixed amount of ligand is between about 0.01 Kd and about
2 Kd.

[0027]In another aspect, the invention provides a method of identifying an
agent that enhances the binding of a PDZ domain to a ligand, by
immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain
on a surface; (b) contacting the immobilized polypeptide with the ligand
in the presence of a test agent and determining the amount of ligand
bound; and, (c) comparing the amount of ligand bound in the presence of
the test agent with the amount of ligand bound by the polypeptide in the
absence of the test agent, wherein at least two-fold greater binding in
the presence of the test agent compared to the absence of the test agent
indicates that the test agent is an agent that enhances the binding of
the PDZ domain to the ligand. In an embodiment, the polypeptide is
immobilized by binding the polypeptide to an immobilized immunoglobulin
that binds the non-PDZ domain.

[0028]In another aspect, the invention provides a method of determining
the potency (Kenhancer) of an enhancer of binding between a PDZ
domain and a ligand, by (a) immobilizing a polypeptide comprising the PDZ
domain and a non-PDZ domain on a surface; (b) contacting the immobilized
polypeptide with a plurality of different mixtures of the ligand and
enhancer, wherein the different mixtures comprise a fixed amount of
ligand, at least a portion of which is detectably labeled, and different
concentrations of the enhancer; (c) determining the amount of ligand
bound at the different concentrations of enhancer; (d) calculating the
potency (Kenhancer) of the enhancer from the binding determined in
(c). In an embodiment, the polypeptide is immobilized by binding the
polypeptide to an immobilized immunoglobulin that binds the non-PDZ
domain. in an embodiment, the fixed amount of ligand is between about
0.01 Kd and about 0.5 Kd.

[0029]In another aspect, the invention provides a method of identifying a
high specificity interaction between a particular PDZ domain and a ligand
known or suspected ofbinding at least one PDZ domain, by (a) providing a
plurality of different immobilized polypeptides, each of said
polypeptides comprising a PDZ domain and a non-PDZ domain; (b)
determining the affinity of the ligand for each of said polypeptides; (c)
comparing the affinity of binding of the ligand to each of said
polypeptides. An interaction between the ligand and a particular PDZ
domain is deemed to have high specificity when the ligand binds an
immobilized polypeptide comprising the particular PDZ domain with at
least 2-fold higher affinity than to immobilized polypeptides not
comprising the particular PDZ domain (a). In an embodiment, the
polypeptide is immobilized by binding the polypeptide to an immobilized
immunoglobulin that binds the non-PDZ domain.

[0030]In another aspect, the invention provides a method for determining
the PDZ-PL inhibition profile of a compound by (a) providing (i) a
plurality of different immobilized polypeptides, each of said
polypeptides comprising a PDZ domain and a non-PDZ domain; (ii) a
plurality of corresponding ligands, wherein each ligand binds at least
one PDZ domain in (i); (b) contacting each of said immobilized
polypeptides in (i) with a corresponding ligand in (ii) in the presence
and absence of a test compound; (c) determining for each
polypeptide-ligand pair in (b) whether the test compound inhibits binding
between the immobilized polypeptide and the corresponding ligand thereby
determining the PDZ-PL inhibition profile of the test compound.

[0031]In another aspect, the invention provides an array comprising a
plurality of different immobilized polypeptides, each of said
polypeptides comprising a PDZ domain and a non-PDZ domain. In an
embodiment, the array is situated in a plastic multiwell plate. In an
embodiment, the array has at least 12 different polypeptides comprising
at least 12 different PDZ domains, for example, at least 12 different PDZ
domains are from PDZs expressed in lymphocytes. In an embodiment, the
PDZs are selected from those listed in Table 2 or 6.

[0032]In an aspect, the invention provides an assay device comprising a
plurality of different immobilized PDZ-containing proteins organized in
an array. In one embodiment, the device has at least 25 different
PDZ-containing proteins.

[0033]In a further aspect, the invention provides a method for identifying
an interaction between a PDZ domain and a PL by contacting a PL to a
plurality of PDZ containing polypeptides and detecting binding of at
least one PL to a PDZ. In an embodiment, the contacting occurs on assay
device comprising a plurality of different immobilized PDZ-containing
proteins organized in an array. In one embodiment, the device has at
least 25 different PDZ-containing proteins. In embodiments, an
interaction between a PDZ and more than one PL, or between a PL and more
than one PDZ, is detected.

[0034]In a related aspect, the invention provides method for identifying a
modulator of an interaction between a PDZ and a PL by conducting any of
the aforementioned assays in the presence and absence of a test compound
and detecting a difference in at least one PDZ-PL interaction in the
presence and absence of the test compound. In embodiments, the modulator
is an enhancer of the interaction. In other embodiments, the modulator is
an inhibitor of the interaction.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIGS. 1A-1D show the results of exemplary assays in which the
binding of biotinylated peptides having a sequence of the
carboxyl-terminus ("c-terminus") of various leukocyte proteins to PDZ
domains (i.e., GST-PDZ domain fusion proteins) was determined using the
"G" assay described infra. The PDZ domains are: PSD95 (FIG. 1A); NeDLG
(FIG. 1B); DLG1 (FIG. 1C); and 41.8 (FIG. 1D). These and other PDZ domain
fusion proteins are described infra (e.g., TABLE 2). In the figure,
peptides 1-31 refer to the biotinylated PL peptide used in the assay, and
are identified in the Key, infra. "Peptide IDs" are defined in TABLE 3.
Key:

[0036]FIGS. 2A and 2B show the Apparent Affinity Determination for
PDZ-Ligand Interactions. Varying concentrations of biotinylated CLASP-2
(FIG. 2A; TABLE 4) or Fas (FIG. 2B; TABLE 4) C-terminal peptides were
reacted with immobilized (plate bound) GST polypeptide or GST-PDZ fusion
proteins (GST-DLG1, GST-NeDLG, and GST-PSD95). The binding to GST alone
(<0.2 OD units) was subtracted from the binding to the fusion proteins
to obtain the signal at each peptide concentration. This signal was then
normalized by dividing the signal at each peptide concentration by the
maximum signal observed for each peptide-PDZ pair (i.e. the signal
obtained at 30 μM CLASP 2 peptide or 100 μM Fas peptide; 0.4-1.0 OD
units for CLASP 2 and 1.2-2.0 OD units for Fas). The normalized signals
were then plotted and fit to a saturation binding curve, yielding an
apparent affinity of 21 μM for DLG1-CLASP 2 interaction, 7.5 μM for
NeDLG-CLASP 2 interaction, 45 μM for PSD95-CLASP 2 interaction, 54
μM for DLG1-Fas interaction, 54 μM for NeDLG-Fas interaction, and
85 μM for PSD95-Fas interaction. Data are means of duplicate data
points, with standard errors between duplicate data points <20%.

[0037]FIGS. 3A-3F show inhibition of PDZ-PL peptide interactions. A fixed
concentration of biotinylated C-terminal peptide having a sequence based
on the C-terminal sequence of a cell surface receptor protein (CLASP 2,
CD46, Fas, and KV1.3; see TABLE 4) was bound to immobilized GST
polypeptide or the GST-fusion protein indicated at the top left of each
frame, in the presence or absence of the competitor peptides indicated in
the legend of each frame and the level of inhibition determined. FIG.
3A-DLG1; FIG. 3B-PSD95; FIG. 3C NeDLG; FIG. 3D-DLG1, FIG. 3E, PSD95; Fig.
F-41.8. In FIG. 3A-B the competitor peptides are present at 100 μM; in
FIGS. 3C-F the competitor is present at the indicated concentration.

[0038]FIGS. 4A and 4B shows the results of introduction of a Tat-CD3
fusion peptide on T cell activation. Antigen-specific T cell activation
was measured by cytokine production. Fusion peptides containing tat and a
T cell surface molecule carboxyl terminus inhibited γ-interferon
(IFN) production by a T cell line in response to myelin basic protein
(MBP) stimulation. The level of inhibition was determined by first
subtracting the binding of the labeled peptide to GST alone from the
binding to the fusion protein and dividing by the signal in the absence
of competitor peptide.

[0039]5.1 A "fusion protein" or "fusion polypeptide" as used herein refers
to a composite protein, i.e., a single contiguous amino acid sequence,
made up of two (or more) distinct, heterologous polypeptides which are
not normally fused together in a single amino acid sequence. Thus, a
fusion protein can include a single amino acid sequence that contains two
entirely distinct amino acid sequences or two similar or identical
polypeptide sequences, provided that these sequences are not normally
found together in the same configuration in a single amino acid sequence
found in nature. Fusion proteins can generally be prepared using either
recombinant nucleic acid methods, i.e., as a result of transcription and
translation of a recombinant gene fusion product, which fusion comprises
a segment encoding a polypeptide of the invention and a segment encoding
a heterologous protein, or by chemical synthesis methods well known in
the art.

[0040]5.2 A "fusion protein construct" as used herein is a polynucleotide
encoding a fusion protein.

[0042]PDZ domains are found in diverse membrane-associated proteins
including members of the MAGUK family of guanylate kinase homologs,
several protein phosphatases and kinases, neuronal nitric oxide synthase,
and several dystrophin-associated proteins, collectively known as
syntrophins.

[0043]Exemplary PDZ domain-containing proteins and PDZ domain sequences
are shown in TABLE 3. The term "PDZ domain" also encompasses variants
(e.g., naturally occurring variants) of the sequences of TABLE 3 (e.g.,
polymorphic variants, variants with conservative substitutions, and the
like). Typically, PDZ domains are substantially identical to those shown
in TABLE 3, e.g., at least about 70%, at least about 80%, or at least
about 90% amino acid residue identity when compared and aligned for
maximum correspondence.

[0045]5.5 As used herein, the term "PDZ-domain polypeptide" refers to a
polypeptide containing a PDZ domain, such as a fusion protein including a
PDZ domain sequence, a naturally occurring PDZ protein, or an isolated
PDZ domain peptide.

[0046]5.6 As used herein, the term "PL protein" or "PDZ Ligand protein"
refers to a naturally occurring protein that forms a molecular complex
with a PDZ-domain, or to a protein whose carboxy-terminus, when expressed
separately from the full length protein (e.g., as a peptide fragment of
4-25 residues, e.g., 16 residues), forms such a molecular complex. The
molecular complex can be observed in vitro using the "A assay" or "G
assay" described infra, or in vivo. Exemplary PL proteins listed in TABLE
2 are demonstrated to bind specific PDZ proteins. This definition is not
intended to include anti-PDZ antibodies and the like.

[0047]5.7 As used herein, a "PL sequence" refers to the amino acid
sequence of the C-terminus of a PL protein (e.g., the C-terminal 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 residues) ("C-terminal PL
sequence") or to an internal sequence known to bind a PDZ domain
("internal PL sequence).

[0048]5.8 As used herein, a "PL peptide" is a peptide of having a sequence
from, or based on, the sequence of the C-terminus of a PL protein.
Exemplary PL peptides (biotinylated) are listed in TABLE 4.

[0049]5.9 As used herein, a "PL fusion protein" is a fusion protein that
has a PL sequence as one domain, typically as the C-terminal domain of
the fusion protein. An exemplary PL fusion protein is a tat-PL sequence
fusion.

[0050]5.10 As used herein, the term "PL inhibitor peptide sequence" refers
to PL peptide an amino acid sequence that (in the form of a peptide or PL
fusion protein) inhibits the interaction between a PDZ domain polypeptide
and a PL peptide (e.g., in an A assay or a G assay).

[0051]5.11 As used herein, a "PDZ-domain encoding sequence" means a
segment of a polynucleotide encoding a PDZ domain. In various
embodiments, the polynucleotide is DNA, RNA, single stranded or double
stranded.

[0052]5.12 As used herein, the terms "antagonist" and "inhibitor," when
used in the context of modulating a binding interaction (such as the
binding of a PDZ domain sequence to a PL sequence), are used
interchangeably and refer to an agent that reduces the binding of the,
e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence
(e.g., PDZ protein, PDZ domain peptide).

[0053]5.13 As used herein, the terms "agonist" and "enhancer," when used
in the context of modulating a binding interaction (such as the binding
of a PDZ domain sequence to a PL sequence), are used interchangeably and
refer to an agent that increases the binding of the, e.g., PL sequence
(e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZ protein,
PDZ domain peptide).

[0054]5.14 As used herein, the terms "peptide mimetic," "peptidomimetic,"
and "peptide analog" are used interchangeably and refer to a synthetic
chemical compound which has substantially the same structural and/or
functional characteristics of an PL inhibitory or PL binding peptide of
the invention. The mimetic can be either entirely composed of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of
partly natural peptide amino acids and partly non-natural analogs of
amino acids. The mimetic can also incorporate any amount of natural amino
acid conservative substitutions as long as such substitutions also do not
substantially alter the mimetic's structure and/or inhibitory or binding
activity. As with polypeptides of the invention which are conservative
variants, routine experimentation will determine whether a mimetic is
within the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Thus, a mimetic composition is
within the scope of the invention if it is capable of binding to a PDZ
domain and/or inhibiting a PL-PDZ interaction.

[0056]A polypeptide can be characterized as a mimetic when all or some of
its residues are joined by chemical means other than natural peptide
bonds. Individual peptidomimetic residues can be joined by peptide bonds,
other chemical bonds or coupling means, such as, e.g., glutaraldehyde,
N-hydroxysuccinimide esters, bifunctional maleimides,
N,N=-dicyclohexylcarbodiimide (DCC) or N,N=-diisopropylcarbodiimide
(DIC). Linking groups that can be an alternative to the traditional amide
bond ("peptide bond") linkages include, e.g., ketomethylene (e.g.,
--C(═O)--CH2-- for --C(═O)--NH--), aminomethylene
(CH2--NH), ethylene, olefin (CH═CH), ether (CH2--O),
thioether (CH2--S), tetrazole (CN4--), thiazole, retroamide,
thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and
Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, A
Peptide Backbone Modifications, Marcell Dekker, N.Y).

[0057]A polypeptide can also be characterized as a mimetic by containing
all or some non-natural residues in place of naturally occurring amino
acid residues. Normatural residues are well described in the scientific
and patent literature; a few exemplary normatural compositions useful as
mimetics of natural amino acid residues and guidelines are described
below.

[0059]Mimetics of acidic amino acids can be generated by substitution by,
e.g., non-carboxylate amino acids while maintaining a negative charge;
(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,
aspartyl or glutamyl) can also be selectively modified by reaction with
carbodiimides (R═N--C--N--R═) such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl
can also be converted to asparaginyl and glutaminyl residues by reaction
with ammonium ions.

[0060]Mimetics of basic amino acids can be generated by substitution with,
e.g., (in addition to lysine and arginine) the amino acids ornithine,
citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid,
where alkyl is defined above. Nitrile derivative (e.g., containing the
CN-moiety in place of COOH) can be substituted for asparagine or
glutamine. Asparaginyl and glutaminyl residues can be deaminated to the
corresponding aspartyl or glutamyl residues.

[0062]Tyrosine residue mimetics can be generated by reacting tyrosyl with,
e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol
and tetranitromethane can be used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively.

[0064]Lysine mimetics can be generated (and amino terminal residues can be
altered) by reacting lysinyl with, e.g., succinic or other carboxylic
acid anhydrides. Lysine and other alpha-amino-containing residue mimetics
can also be generated by reaction with imidoesters, such as methyl
picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and
transamidase-catalyzed reactions with glyoxylate.

[0066]Other mimetics include, e.g., those generated by hydroxylation of
proline and lysine; phosphorylation of the hydroxyl groups of seryl or
threonyl residues; methylation of the alpha-amino groups of lysine,
arginine and histidine; acetylation of the N-terminal amine; methylation
of main chain amide residues or substitution with N-methyl amino acids;
or amidation of C-terminal carboxyl groups.

A component of a natural polypeptide (e.g., a PL polypeptide or PDZ
polypetide) can also be replaced by an amino acid (or peptidomimetic
residue) of the opposite chirality. Thus, any amino acid naturally
occurring in the L-configuration (which can also be referred to as the R
or S, depending upon the structure of the chemical entity) can be
replaced with the amino acid of the same chemical structural type or a
peptidomimetic, but of the opposite chirality, generally referred to as
the D-amino acid, but which can additionally be referred to as the R-- or
S-- form.

[0068]5.15 As used herein, "peptide variants" and "conservative amino acid
substitutions" refer to peptides that differ from a reference peptide
(e.g., a peptide having the sequence of the carboxy-terminus of a
specified PL protein) by substitution of an amino acid residue having
similar properties (based on size, polarity, hydrophobicity, and the
like). Thus, insofar as the compounds that are encompassed within the
scope of the invention are partially defined in terms of amino acid
residues of designated classes, the amino acids may be generally
categorized into three main classes: hydrophilic amino acids, hydrophobic
amino acids and cysteine-like amino acids, depending primarily on the
characteristics of the amino acid side chain. These main classes may be
further divided into subclasses. Hydrophilic amino acids include amino
acids having acidic, basic or polar side chains and hydrophobic amino
acids include amino acids having aromatic or apolar side chains. Apolar
amino acids may be further subdivided to include, among others, aliphatic
amino acids. The definitions of the classes of amino acids as used herein
are as follows:

[0069]"Hydrophobic Amino Acid" refers to an amino acid having a side chain
that is uncharged at physiological pH and that is repelled by aqueous
solution. Examples of genetically encoded hydrophobic amino acids include
Ile, Leu and Val. Examples of non-genetically encoded hydrophobic amino
acids include t-BuA.

[0070]"Aromatic Amino Acid" refers to a hydrophobic amino acid having a
side chain containing at least one ring having a conjugated π-electron
system (aromatic group). The aromatic group may be further substituted
with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro
and amino groups, as well as others. Examples of genetically encoded
aromatic amino acids include Phe, Tyr and Trp. Commonly encountered
non-genetically encoded aromatic amino acids include phenylglycine,
2-naphthylalanine, β-2-thienylalanine,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chloro-phenylalanine,
2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.

[0071]"Apolar Amino Acid" refers to a hydrophobic amino acid having a side
chain that is generally uncharged at physiological pH and that is not
polar. Examples of genetically encoded apolar amino acids include Gly,
Pro and Met. Examples of non-encoded apolar amino acids include Cha.

[0073]"Hydrophilic Amino Acid" refers to an amino acid having a side chain
that is attracted by aqueous solution. Examples of genetically encoded
hydrophilic amino acids include Ser and Lys. Examples of non-encoded
hydrophilic amino acids include Cit and hCys.

[0074]"Acidic Amino Acid" refers to a hydrophilic amino acid having a side
chain pK value of less than 7. Acidic amino acids typically have
negatively charged side chains at physiological pH due to loss of a
hydrogen ion. Examples of genetically encoded acidic amino acids include
Asp and Glu.

[0076]"Polar Amino Acid" refers to a hydrophilic amino acid having a side
chain that is uncharged at physiological pH, but which has a bond in
which the pair of electrons shared in common by two atoms is held more
closely by one of the atoms. Examples of genetically encoded polar amino
acids include Asx and Glx. Examples of non-genetically encoded polar
amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.

[0077]"Cysteine-Like Amino Acid" refers to an amino acid having a side
chain capable of forming a covalent linkage with a side chain of another
amino acid residue, such as a disulfide linkage. Typically, cysteine-like
amino acids generally have a side chain containing at least one thiol
(SH) group. Examples of genetically encoded cysteine-like amino acids
include Cys. Examples of non-genetically encoded cysteine-like amino
acids include homocysteine and penicillamine.

[0078]As will be appreciated by those having skill in the art, the above
classification are not absolute--several amino acids exhibit more than
one characteristic property, and can therefore be included in more than
one category. For example, tyrosine has both an aromatic ring and a polar
hydroxyl group. Thus, tyrosine has dual properties and can be included in
both the aromatic and polar categories. Similarly, in addition to being
able to form disulfide linkages, cysteine also has apolar character.
Thus, while not strictly classified as a hydrophobic or apolar amino
acid, in many instances cysteine can be used to confer hydrophobicity to
a peptide.

[0080]The classifications of the above-described genetically encoded and
non-encoded amino acids are summarized in TABLE 1, below. It is to be
understood that TABLE 1 is for illustrative purposes only and does not
purport to be an exhaustive list of amino acid residues which may
comprise the peptides and peptide analogues described herein. Other amino
acid residues which are useful for making the peptides and peptide
analogues described herein can be found, e.g., in Fasman, 1989, CRC
Practical Handbook of Biochemistry and Molecular Biology, CRC Press,
Inc., and the references cited therein. Amino acids not specifically
mentioned herein can be conveniently classified into the above-described
categories on the basis of known behavior and/or their characteristic
chemical and/or physical properties as compared with amino acids
specifically identified.

[0081]5.16 As used herein, a "detectable label" has the ordinary meaning
in the art and refers to an atom (e.g., radionuclide), molecule (e.g.,
fluorescein), or complex, that is or can be used to detect (e.g., due to
a physical or chemical property), indicate the presence of a molecule or
to enable binding of another molecule to which it is covalently bound or
otherwise associated. The term "label" also refers to covalently bound or
otherwise associated molecules (e.g., a biomolecule such as an enzyme)
that act on a substrate to produce a detectable atom, molecule or
complex. Detectable labels suitable for use in the present invention
include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Labels useful in the present invention include biotin for staining with
labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads®),
fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green
fluorescent protein, enhanced green fluorescent protein, and the like),
radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P),
enzymes (e.g., hydrolases, particularly phosphatases such as alkaline
phosphatase, esterases and glycosidases, or oxidoreductases, particularly
peroxidases such as horse radish peroxidase, and others commonly used in
ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups,
chromogenic agents, and colorimetric labels such as colloidal gold or
colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents teaching the use of such labels include U.S. Pat. Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241. Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels and chemiluminescent
labels may be detected using photographic film or scintillation counters,
fluorescent markers may be detected using a photodetector to detect
emitted light (e.g., as in fluorescence-activated cell sorting).
Enzymatic labels are typically detected by providing the enzyme with a
substrate and detecting the reaction product produced by the action of
the enzyme on the substrate, and colorimetric labels are detected by
simply visualizing the colored label. Thus, a label is any composition
detectable by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. The label may be coupled directly
or indirectly to the desired component of the assay according to methods
well known in the art. Non-radioactive labels are often attached by
indirect means. Generally, a ligand molecule (e.g., biotin) is covalently
bound to the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal generating system, such as a detectable
enzyme, a fluorescent compound, or a chemiluminescent compound. A number
of ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used
in conjunction with the labeled, naturally occurring anti-ligands.
Alternatively, any haptenic or antigenic compound can be used in
combination with an antibody. The molecules can also be conjugated
directly to signal generating compounds, e.g., by conjugation with an
enzyme or fluorophore. Means of detecting labels are well known to those
of skill in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter, photographic
film as in autoradiography, or storage phosphor imaging. Where the label
is a fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the resulting
fluorescence. The fluorescence may be detected visually, by means of
photographic film, by the use of electronic detectors such as charge
coupled devices (CCDs) or photomultipliers and the like. Similarly,
enzymatic labels may be detected by providing the appropriate substrates
for the enzyme and detecting the resulting reaction product. Also, simple
calorimetric labels may be detected by observing the color associated
with the label. It will be appreciated that when pairs of fluorophores
are used in an assay, it is often preferred that they have distinct
emission patterns (wavelengths) so that they can be easily distinguished.

[0082]5.17 As used herein, the term "substantially identical" in the
context of comparing amino acid sequences, means that the sequences have
at least about 70%, at least about 80%, or at least about 90% amino acid
residue identity when compared and aligned for maximum correspondence. An
algorithm that is suitable for determining percent sequence identity and
sequence similarity is the FASTA algorithm, which is described in
Pearson, W. R. & Lipman, D. J., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:
2444. See also W. R. Pearson, 1996, Methods Enzymol. 266: 227-258.
Preferred parameters used in a FASTA alignment of DNA sequences to
calculate percent identity are optimized, BL50 Matrix 15: -5, k-tuple=2;
joining penalty=40, optimization=28; gap penalty=-12, gap length
penalty=-2; and width=16.

[0085]5.20 The term "specific binding" refers to binding between two
molecules, for example, a ligand and a receptor, characterized by the
ability of a molecule (ligand) to associate with another specific
molecule (receptor) even in the presence of many other diverse molecules,
i.e., to show preferential binding of one molecule for another in a
heterogeneous mixture of molecules. Specific binding of a ligand to a
receptor is also evidenced by reduced binding of a detectably labeled
ligand to the receptor in the presence of excess unlabeled ligand (i.e.,
a binding competition assay).

[0086]5.21 As used herein, a "plurality" of PDZ proteins (or corresponding
PDZ domains or PDZ fusion polypeptides) has its usual meaning. In some
embodiments, the plurality is at least 5, and often at least 25, at least
40 or at least 60 different PDZ proteins. In some embodiments, the
plurality is selected from the list of PDZ polypeptides listed in Table 2
or Table 6. In some embodiments, the plurality of different PDZ proteins
are from (i.e., expressed in) a particular specified tissue or a
particular class or type of cell. In some embodiments, the plurality of
different PDZ proteins represents a substantial fraction (e.g., typically
at least 50%, more often at least 80%) of all of the PDZ proteins known
to be, or suspected of being, expressed in the tissue or cell(s), e.g.,
all of the PDZ proteins known to be present in lymphocytes or
hematopoetic cells. In some embodiments, the plurality is at least 50%,
usually at least 80%, at least 90% or all of the PDZ proteins disclosed
herein as being expressed in hematopoietic cells (see Tables 2 and 6). I
an embodiment, the plurality includes at least 1, often at least 2,
sometimes at least 5 or at least 10 and sometimes all of the following
PDZ proteins: BAI I associated prot., Connector enhancer, DLG5 (pdlg),
DVL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot.,
KIAA147, KIAA0300, KIAA0380, KIAA0440, KIAA0545, KIAA0807, KIAA0858,
KIAA0902, novel serine protease, PDZK1, PICK8, PTN-3, RPIP8, serine
protease, 26s subunit p27, hSYNTENIN, TAX1-IP, TAX2-like protein, wwp3,
X11 prot. beta, ZO1. When referring to PL ligands or corresponding PL
proteins (e.g., corresponding to those listed in Table 2, Table 4, Table
5, or elsewhere herein) a "plurality" may refer to at least 5, at least
10, and often at least 25 PLs such as those specifically listed herein,
or to the classes and percentages set forth supra for PDZ domains.

6. DETAILED DESCRIPTION OF THE INVENTION

[0087]The present inventors have discovered that interactions between PDZ
proteins and PL proteins play an important and extensive role in the
biological function of hematopoietic cells and other cells involved in
the immune response. Although PDZ-PL interactions were known in the
nervous system (i.e., in neurons), their universal importance in
hematopoietic cell function, especially in function of T cells and B
cells, and their fundamental role in modulation of the immune response
has not been recognized. In particular, the present inventors have
surprisingly discovered that cell adhesion molecules that mediate
cell-cell interaction in the hematopoietic system are PDZ-binding
proteins (PL proteins) and bind to PDZ proteins. The inventors have
identified numerous interactions between PDZ proteins and PL proteins
present in immune system cells, and the invention provides reagents and
methods for affecting biological function in the immune system by
inhibiting these interactions. As used herein, the term "biological
function" in the context of a cell, refers to a detectable biological
activity normally carried out by the cell, e.g., a phenotypic change such
as proliferation, cell activation (e.g., T cell activation, B cell
activation, T-B cell conjugate formation), cytokine release,
degranulation, tyrosine phosphorylation, ion (e.g., calcium) flux,
metabolic activity, apoptosis, changes in gene expression, maintenance of
cell structure, cell migration, adherence to a substrate, signal
transduction, cell-cell interactions, and others described herein or
known in the art.

[0088]In one aspect, the present invention relates to peptides, peptide
analogues or mimetics, pharmaceutical compositions, and methods of using
such compositions to regulate the biological activities of hematopoietic
cells, e.g. T cells and B cells, or other cells (e.g., endothelial cells)
that necessary for immune function. The invention further relates to
methods of using the compositions to modulate hematopoietic cell
activation and immune function, as well as assays for such inhibitors.

[0089]TABLE 2 summarizes interaction profiles for an extensive analysis of
protein interactions in T cells and B cells. PDZ proteins, the vast
majority of which were not previously known to be expressed in immune
system cells, are listed in the top row of TABLE 2. The first column of
the table lists PL proteins. Positions in the matrix denoted by the
letter "A" or "G" indicate that an interaction between the PDZ protein
and the PL has been detected in novel binding assays (described in detail
in, e.g., Section 6.2, infra). A blank cell indicates that no interaction
was detected using the assays of the invention. An asterisk (*) denotes a
PL-PDZ interaction previously reported in the scientific literature.

[0091]As discussed in detail herein, the PDZ proteins listed in TABLE 2
are naturally occurring proteins containing a PDZ domain. The present
invention is particularly directed to the detection and modulation of
interactions between PDZ proteins and PL proteins in hematopoietic cells.
Exemplary PL proteins are listed in TABLE 2. Notably, as discussed infra,
many of these PL proteins have not previously been recognized as such in
any cell system. A variety of PL protein classes are known, and the PL
proteins described herein can be characterized as (1) "PL adhesion
proteins" (2) "PL ion channel proteins" (3) "PL adaptor proteins" (4) "PL
intracellular proteins" and (5) "PL cytokine receptor proteins."

[0092]As used herein, an adhesion protein is a cell surface protein
involved in cell-cell interaction by direct contact with cell surface
molecules (e.g., transmembrane proteins or surface proteins) on a
different cell. Thus, when a cell expressing a PL adhesion protein
contacts an appropriate other cell, the PL adhesion protein localizes at
the interface of the two cells and directly contacts a cell surface
molecule on the second cell. A cell-cell interface is a region where the
plasma membranes of two different cells are in close (generally <10
nm, often about 1 nm) apposition. Typically, direct molecular contact
means interaction of molecules at distances where Van der Walls forces
are significant, generally less than about 1 nm. Exemplary PL adhesion
proteins include CD6; CD49E (alpha-4); CD49F (a form, alpha6); CD138
(syndecan); CLASP-1; CLASP-4; VCAM1; CLASP-2; DNAM-1; CD83; CD44 (long
form); CD97; (CD55L); CD3n; DOCK2; CD34; and FceRIb. Thus, in one
embodiment, the PL proteins of the invention are PL adhesion proteins. In
an embodiment, the invention provides methods and reagents, as detailed
herein, for inhibiting interactions between PL adhesion proteins and PDZ
proteins to modulate an immune response. In an embodiment, the inhibition
or modulation occurs in a hematopoietic cell. In a related embodiment,
the inhibition or modulation occurs in an endothelial cell. In a related
embodiment, the inhibition or modulation occurs in an endothelial cell.
In a related embodiment, the inhibition or modulation occurs in an
epithelial cells, keratinocytes, hepatocytes, cardiac myocytes.

[0093]As used herein, an ion channel protein means a transmembrane protein
that itself catalyzes the passage of an ion from aqueous solution on one
side of a lipid bilayer membrane to aqueous solution on the other side
(e.g., by forming a small pore in the membrane). One exemplary PL ion
channel proteins is Kv1.3. Thus, in one embodiment, the PL proteins of
the invention are PL ion channel proteins. In an embodiment, the
invention provides methods and reagents, as detailed herein, for
inhibiting interactions between PL ion channel proteins and PDZ proteins
to modulate an immune response. In an embodiment, the inhibition or
modulation occurs in a hematopoeitic cell. In a related embodiment, the
inhibition or modulation occurs in an endothelial cell.

[0094]As used herein, an intercellular (i.e., cytosolic) protein has the
normal meaning in the art and refers to a protein that is not membrane
bound, e.g., has no transmembrane domain. Thus, in one embodiment, the PL
proteins of the invention are PL intercellular proteins. Exemplary PL
intercellular proteins include Glycophorin C and LPAP. In an embodiment,
the invention provides methods and reagents, as detailed herein, for
inhibiting interactions between PL cytoplasmic proteins and PDZ proteins
to modulate an immune response. In an embodiment, the inhibition or
modulation occurs in a hematopoeitic cell. In a related embodiment, the
inhibition or modulation occurs in an endothelial cell.

[0095]As used herein a cytokine receptor has the normal meaning in the art
and refers to a membrane protein with an extracellular domain that
specifically binds a cytokine. Exemplary PL cytokine receptor proteins
include CDW125 (IL5R), CDW128A (IL8RA), and BRL-1. Thus, in one
embodiment, the PL proteins of the invention are PL cytokine proteins. In
an embodiment, the invention provides methods and reagents, as detailed
herein, for inhibiting interactions between PL cytokine proteins and PDZ
proteins to modulate an immune response. In an embodiment, the inhibition
or modulation occurs in a hematopoeitic cell. In a related embodiment,
the inhibition or modulation occurs in an endothelial cell.

[0096]As used herein, an adaptor protein means a molecule (e.g., protein)
that contributes to the formation of a multimolecular complex by binding
two or more other biomolecuics. The binding of the two or more other
molecules by the adaptor molecule/protein generally involves direct
molecular contact between the adaptor protein and each of the two or more
other moiccules. One exemplary PL adaptor protein is LPAP. Thus, in one
embodiment, the PL proteins of the invention are PL adaptor proteins. In
an embodiment, the invention provides methods and reagents, as detailed
herein, for inhibiting interactions between PL adaptor proteins and PDZ
proteins to modulate an immune response. In an embodiment, the inhibition
or modulation occurs in a hematopoeitic cell. In a related embodiment,
the inhibition or modulation occurs in an endothelial cell.

[0097]In various embodiments, the PL proteins of the invention are
characterized by specific C-terminal (i.e., PL domain) amino acid
sequences or amino acid motifs, as described elsewhere in this
disclosure.

[0098]In various embodiments of the invention, the PL proteins of the
invention bind a PDZ protein expressed in T lymphocytes, B lymphocytes,
or both T and B lymphocytes. In an embodiment, the PL protein binds a PDZ
protein expressed in endothelial cells. In various embodiments, the PL
proteins and/or the PDZ protein to which it binds are not expressed in
the nervous system (e.g., neurons).

[0099]In various embodiments of the invention, the PL protein of the
invention binds only one PDZ protein listed in TABLE 2. In other
embodiment, the PL protein binds 1 to 3, 3 to 5, or more than 5 different
PDZ proteins listed in TABLE 2.

[0100]In various embodiments of the invention, the PL protein is expressed
or up-regulated upon cell activation (e.g., in activated B lymphocytes, T
lymphocytes) or upon entry into mitosis (e.g., up-regulation in rapidly
proliferating cell populations).

[0101]In various embodiments of the invention, the PL protein is (i) a
protein that mediates immune cell (e.g., hematopoietic cell) activation
or migration, (ii) a protein that does not mediate apoptosis in a cell
type (iii) a protein that is other than a G-protein coupled seven
transmembrane helix receptor, (iv) a protein that is G-protein coupled
seven transmembrane helix receptor but not a cytokine receptor or (v) a
protein that is not a G-protein coupled seven transmembrane helix
receptor and is a cytokine receptor.

[0102]As noted supra, the present inventors surprisingly discovered that
numerous PDZ proteins are expressed in immune system cells, and play a
fundamental biological role in modulation of the immune response. PDZ
proteins DLG1 and TIAM-1 have been previously described to be in T cells.
The present inventors discovered, using a BLAST search of the Human EST
database and the experiments described infra, that several additional PDZ
proteins are present in hematopoietic cells including MPP1, P-DLG,
VELI-1, PSD95, syntenin in T cells and CASK, DLG1, DLG2, ZIP KINASE,
syntrophin 2, P-dlg, PSD95, and syntenin in B cells.

[0103]To determine the full extent of PDZ proteins' involvement in
hematopoietic function, the inventors embarked on a systematic
investigation of PDZ proteins in T and B cells. A comprehensive list of
PDZ domain-containing proteins was retrieved from the Sanger Centre
database (Pfam) searching for the keyword, PDZ. The corresponding cDNA
sequences were retrieved from GenBank using the NCBI "entrez" database
(hereinafter, "GenBank PDZ protein cDNA sequences"). The DNA portion
encoding PDZ domains was identified by alignment of cDNA and protein
sequence using CLUSTALW. Based on the DNA/protein alignment information,
primers encompassing the PDZ domains were designed. The expression of
certain PDZ-containing proteins in immune cells was detected by
polymerase chain reaction ("PCR") amplification of cDNAs obtained by
reverse transcription ("RT") of immune cell derived RNA (i.e., "RT-PCR").
PCR, RT-PCR and other methods for analysis and manipulation of nucleic
acids are well known and are described generally in Sambrook et al.,
(1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2 ND ED., VOLS. 1-3, Cold
Spring Harbor Laboratory hereinafter, "Sambrook"); and Ausubel et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and
Wiley-Interscience, New York (1997), as supplemented through January 1999
(hereinafter "Ausubel").

[0104]In the experiments summarized in TABLE 2, T-cells (Jurkat E6 cell
line) and B-cells (MV 4-11 cell line) were tested for expression of
specific PDZ domain containing genes by RT-PCR. RNA was prepared using
the "trizol" RNA preparation kit (GIBCO-BRL; Cat. # 15596-018) according
to the manufacturer's recommendations. Briefly, 1-5×107
lymphoblasts were harvested by centrifugation at 200×g for 10
minutes at 20° C. Cells were resuspended in 100 μl PBS buffer
and 1 ml of TRIZOL reagent was added per 5×106 cells. The
cells resuspension was mixed and after 5 minutes incubation at room
temperature (RT), chloroform was added at 0.2 ml per ml TRIZOL. The
resuspension was vigorously shaken and incubated for 3 more minutes at
RT. Samples were then centrifuged at 12000×g for 15 minutes at
4° C., the aqueous phase was recovered and RNA was precipitated
with 2-propanol. The precipitate was collected by centrifugation at
12000×g for 15 minutes at 4° C., washed with 75% ethanol,
finally recollected by another spin at 12000×g for 15 minutes at
4° C., air dried and resuspended in an appropriate volume of DEPC
treated water.

[0105]RNA concentration and purity were determined by the measurement of
260/280 nm light absorption by the nucleic acid. For cDNA synthesis, the
SUPERSCRIPT II reverse transcriptase cDNA kit (GIBCO-BRL; Cat. #
18064-014) was used. RNA input per 200 μl cDNA reaction sample was 10
μg. Prior to cDNA synthesis RNA was treated with 1 unit/μl DNAse I
in 110 μl water at 37° C. for 20 minutes. DNase I was then
inactivated by a 10 minutes incubation at 70° C. Random primer was
used for cDNA priming; 10 μl of random hexamer primer (100 ng/μl)
was added, samples were heated to 70° C. for 5 minutes and chilled
on ice. Subsequently 40 μl SUPERSCRIPT II "first strand" buffer, 20
μl of 0.1 M DDT, 10 μl of a 10 mM of mix of deoxynucleotide
triphosphates (dATP, dCTP, dGTP, dUTP) and 10 μl of SUPERSCRIPT II
reverse transcriptase were added and cDNA synthesis was done for 45
minutes at 42° C. Reactions were stopped by a 5 minutes incubation
at 95° C. and typically, 2-4 μl of such cDNA samples were used
for PCR.

[0106]A portion of the cDNA (typically, 1/5 of a 20 μl reaction) was
used for PCR. PCR was conducted using primers designed to amplify
specifically PDZ domain-containing regions of PDZ proteins of interest.
Oligonucleotide primers were designed to amplify one or more PDZ-encoding
domains. The DNA sequences encoding the various PDZ domains of interest
were identified by inspection (i.e., conceptual translation of the PDZ
protein cDNA sequences obtained from GenBank, followed by alignment with
the PDZ domain amino acid sequence). TABLE 3 shows the PCR primers, the
PDZ-encoded domains amplified, and the GenBank accession number of the
PDZ-domain containing proteins. To facilitate subsequent cloning of PDZ
domains, the PCR primers included endonuclease restriction sequences at
their ends to allow ligation with pGEX-3X cloning vector (Pharmacia,
GenBank XXI13852) in frame with glutathione-S transferase (GST).

[0109]Two complementary assays, termed "A" and "G" were developed to
detect binding between a PDZ-domain polypeptide and candidate PDZ ligand.
In each of the two different assays, binding, is detected between a
peptide having a sequence corresponding to the C-terminus of a protein
anticipated to bind to one or more PDZ domain (i.e. a candidate PL
peptide) and a PDZ-domain polypeptide (typically a fusion protein
containing a PDZ domain). In the "A" assay, the candidate, PL peptide is
immobilized and binding of a soluble PDZ-domain polypeptide to the
immobilized peptide is detected (the "A" assay is named for the fact that
in one embodiment an avidin surface is used to immobilize the peptide).
In the "G" assay, the PDZ-domain polypeptide is immobilized and binding
of a soluble PL peptide is detected (The "G" assay is named for the fact
that in one embodiment a GST-binding surface is used to immobilize the
PDZ-domain polypeptide). Preferred embodiments of these assays are
described in detail infra. However, it will be appreciated by ordinarily
skilled practitioners that these assays can be modified in numerous ways
while remaining useful for the purposes of the present invention.

[0110]6.2.1 Production of Fusion Proteins Containing PDZ-Domains

[0111]GST-PDZ domain fusion proteins were prepared for use in the assays
of the invention. PCR products containing PDZ encoding domains (as
described in §6.1 supra) were subcloned into an expression vector to
permit expression of fusion proteins containing a PDZ domain and a
heterologous domain (i.e., a glutathione-S transferase sequence, "GST").
PCR products (i.e., DNA fragments) representing PDZ domain encoding DNA
was extracted from agarose gels using the "sephaglas" gel extraction
system (Pharmacia) according to the manufacturer's recommendations.

[0112]As noted supra, PCR primers were designed to include endonuclease
restriction sites to facilitate ligation of PCR fragments into a GST gene
fusion vector (pGEX-3X; Pharmacia, GenBank accession no. XXU13852)
in-frame with the glutathione-S transferase coding sequence. This vector
contains a IPTG inducible lacZ promoter. The pGEX-3X vector was
linearized using Bam HI and Eco RI or, in some cases, Eco RI or Sma I, as
shown in TABLE 3, and dephosphorylated. For most cloning approaches,
double digest with Bam HI and Eco RI was performed, so that the ends of
the PCR fragments to clone were Bam HI and Eco RI. In some cases,
restriction endonuclease combinations used were Bgl II and Eco RI, Bam HI
and Mfe I, or Eco RI only, Sma I only, or BamHI only (see TABLE 3). When
more than one PDZ domain was cloned, the DNA portion cloned represents
the PDZ domains and the cDNA portion located between individual domains.
Precise locations of cloned fragments used in the assays are indicated in
TABLE 3. DNA linker sequences between the GST portion and the PDZ domain
containing DNA portion vary slightly, dependent on which of the above
described cloning sites and approaches were used. As a consequence, the
amino acid sequence of the GST-PDZ fusion protein varies in the linker
region between GST and PDZ domain. Protein linkers sequences
corresponding to different cloning sites/approaches are shown below.
Linker sequences (vector DNA encoded) are bold, PDZ domain containing
gene derived sequences are in italics. [0113]1) GST-BamHI/BamH--PDZ
domain insert Gly-IIe-PDZ domain insert [0114]2) GST-BamHI/BglII--PDZ
domain insert Gly-Ile-domain insert [0115]3) GST-EcoRI/EcoI--PDZ domain
insert Gly-Ile-ProGly-Asn--PDZ domain insert (SEQ ID NO:258) [0116]4)
GST-SmaI/SmaI--PDZ domain insert Gly-Ile-Pro--PDZ domain insert

[0117]The PDZ-encoding PCR fragment and linearized pGEX-3X vector were
ethanol precipitated and resuspended in 10 μl standard ligation
buffer. Ligation was performed for 4-10 hours at 7° C. using T4
DNA ligase. It will be understood that some of the resulting constructs
include very short linker sequences and that, when multiple PDZ domains
were cloned, the constructs included some DNA located between individual
PDZ domains.

[0118]The ligation products were transformed in DH5a or BL-21 E. coli
bacteria strains. Colonies were screened for presence and identity of the
cloned PDZ domain containing DNA as well as for correct fusion with the
glutathione S-transferase encoding DNA portion by PCR and by sequence
analysis. Positive clones were tested in a small scale assay for
expression of the GST/PDZ domain fusion protein and, if expressing, these
clones were subsequently grown up for large scale preparations of GST/PDZ
fusion protein.

[0119]GST-PDZ domain fusion protein was overexpressed following addition
of IPTG to the culture medium and purified. Detailed procedure of small
scale and large scale fusion protein expression and purification are
described in "GST Gene Fusion System" (second edition, revision 2;
published by Pharmacia). In brief, a small culture (3-5 mls) containing a
bacterial strain (DH5α, BL21 or JM109) with the fusion protein
construct was grown overnight in LB-media at 37° C. with the
appropriate antibiotic selection (100 ug/ml ampicillin; a.k.a. LB-amp).
The overnight culture was poured into a fresh preparation of LB-amp
(typically 250-500 mls) and grown until the optical density (OD) of the
culture was between 0.5 and 0.9 (approximately 2.5 hours). IPTG
(isopropyl β-D-thiogalactopyranoside) was added to a final
concentration of 1.0 mM to induce production of GST fusion protein, and
culture was grown an additional 1.5-2.5 hours. Bacteria were collect by
centrifugation (4500 g) and resuspended in Buffer A--(50 mM Tris, pH 8.0,
50 mM dextrose, 1 mM EDTA, 200 uM phenylmethylsulfonylfluoride). An equal
volume of Buffer A+(Buffer A-, 4 mg/ml lysozyme) was added and incubated
on ice for 3 min to lyse bacteria. An equal volume of Buffer B (10 mM
Tris, pH 8.0, 50 mM KCl, 1 mM EDTA. 0.5% Tween-20, 0.5% NP40 (a.k.a.
IGEPAL CA-630), 200 uM phenylmethylsulfonylfluoride) was added and
incubated for an additional 20 min. The bacterial cell lysate was
centrifuged (×20,000 g), and supernatant was added to glutathione
sepharose 4B (Pharmacia, cat no. 17-0765-01) previous swelled
(rehydrated) in 1× phosphate-buffered saline (PBS). The
supernatant-sepharose slurry was poured into a column and washed with at
least 20 bed volumes of 1×PBS. GST fusion protein was eluted off
the glutathione sepharose by applying 0.5-1.0 ml aliquots of 5 mM
glutathione and collected as separate fractions. Concentrations of
fractions were determined using BioRad Protein Assay (cat no. 500-0006)
according to manufacturer's specifications. Those fractions containing
the highest concentration of fusion protein were pooled and dialyzed
against 1×PBS/35% glycerol. Fusion proteins were assayed for size
and quality by SDS gel electrophoresis (PAGE) as described in "Sambrook."
Fusion protein aliquots were stored at minus 80° C. and at minus
20° C.

[0120]6.2.2 Identification of Candidate PL Proteins and Synthesis of
Peptides

[0121]In some non-hematopoietic cells (e.g., neurons, epithelial cells),
certain PDZ domains are known to be bound by the C-terminal residues of
PDZ-binding proteins. To identify PL proteins that function in
hematopoietic and endothelial cells, cell surface receptor proteins were
identified and peptides having the sequence corresponding to the
c-terminus of each protein were synthesized. TABLE 4 lists these
proteins, and provides corresponding C-terminal sequences and GenBank
accession numbers. "CLASP 1" is described in WO 00/20434 (published 13
Apr. 2000). "CLASP 2" and "CLASP 4" are described in copending
applications U.S. Ser. No. 09/547,276 (attorney docket No. 20054-000200)
and 60/196,527 (attorney docket No. 20054-000400), both filed Apr. 11,
2000.

[0122]Synthetic peptides of defined sequence (e.g., corresponding to the
carboxyl-termini of the indicated proteins) can be synthesized by any
standard resin-based method (see, e.g., U.S. Pat. No. 4,108,846; see
also, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223;
Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et
al., 1995, Science 269:202). The peptides used in the assays described
herein were prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth.
Enz. 289:67-83; Wellings and Atherton, 1997, Meth. Enz. 289:44-67). In
some cases (e.g., for use in the A and G assays of the invention),
peptides were labeled with biotin at the amino-terminus by reaction with
a four-fold excess of biotin methyl ester in dimethylsulfoxide with a
catalytic amount of base. The peptides were cleaved from the resin using
a halide containing acid (e.g. trifluoroacetic acid) in the presence of
appropriate antioxidants (e.g. ethanedithiol) and excess solvent
lyophilized.

[0123]Following lyophilization, peptides can be redissolved and purified
by reverse phase high performance liquid chromatography (HPLC). One
appropriate HPLC solvent system involves a Vydac C-18 semi-preparative
column running at 5 mL per minute with increasing quantities of
acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of water
plus 0.1% trifluoroacetic acid. After HPLC purification, the identities
of the peptides are confirmed by MALDI cation-mode mass spectrometry. As
noted, exemplary biotinylated peptides are provided in TABLE 4.

[0124]6.2.3 Detecting PDZ-PL Interactions

[0125]Based on the determination that immune system cells contain both
many PDZ proteins and similarly many candidate PL proteins, it was
apparent to the inventors that characterization of the specific PDZ-PL
interactions among these proteins would require reliable and rapid assays
for such interactions. A variety of assay formats known in the art can be
used to select ligands that are specifically reactive with a particular
protein. For example, solid-phase ELISA immunoassays,
immunoprecipitation, Biacore, and Western blot assays can be used to
identify peptides that specifically bind PDZ-domain polypeptides. As
discussed supra, two different, complementary assays were developed to
detect PDZ-PL interactions. In each, one binding partner of a PDZ-PL pair
is immobilized, and the ability of the second binding partner to bind is
determined. These assays, which are described infra, can be readily used
to screen for hundreds to thousand of potential PDZ-ligand interactions
in a few hours. Thus these assays can be used to identify yet more novel
PDZ-PL interactions in hematopoietic cells. In addition, they can be used
to identify antagonists of PDZ-PL interactions (see infra). In various
embodiments, fusion proteins are used in the assays and devices of the
invention. Methods for constructing and expressing fusion proteins are
well known. Fusion proteins generally are described in Ausubel et al.,
supra, Kroll et al., 1993, DNA Cell. Biol. 12:441, and Imai et al., 1997,
Cell 91:521-30. Usually, the fusion protein includes a domain to
facilitate immobilization of the protein to a solid substrate ("an
immobilization domain"). Often, the immobilization domain includes an
epitope tag (i.e., a sequence recognized by a antibody, typically a
monoclonal antibody) such as polyhistidine (Bush et al, 1991, J. Biol
Chem 266:13811-14), SEAP (Berger et al, 1988, Gene 66:1-10), or M1 and M2
flag (see, e.g., U.S. Pat. Nos. 5,011,912; 4,851,341; 4,703,004;
4,782,137). In an embodiment, the immobilization domain is a GST coding
region. It will be recognized that, in addition to the PDZ-domain and the
particular residues bound by an immobilized antibody, protein A, or
otherwise contacted with the surface, the protein (e.g., fusion protein),
will contain additional residues. In some embodiments these are residues
naturally associated with the PDZ-domain (i.e., in a particular
PDZ-protein) but they may include residues of synthetic (e.g.,
poly(alanine)) or heterologous origin (e.g., spacers of, e.g., between 10
and 300 residues).

[0126]PDZ domain-containing polypeptide used in the methods of the
invention (e.g., PDZ fusion proteins) of the invention are typically made
by (1) constructing a vector (e.g., plasmid, phage or phagemid)
comprising a polynucleotide sequence encoding the desired polypeptide,
(2) introducing the vector into a suitable expression system (e.g., a
prokaryotic, insect, mammalian, or cell free expression system), (3)
expressing the fusion protein and (4) optionally purifying the fusion
protein.

[0127](1) In one embodiment, expression of the protein comprises inserting
the coding sequence into an appropriate expression vector (i.e., a vector
that contains the necessary elements for the transcription and
translation of the inserted coding sequence required for the expression
system employed, e.g., control elements including enhancers, promoters,
transcription terminators, origins of replication, a suitable initiation
codon (e.g., methionine), open reading frame, and translational
regulatory signals (e.g., a ribosome binding site, a termination codon
and a polyadenylation sequence. Depending on the vector system and host
utilized, any number of suitable transcription and translation elements,
including constitutive and inducible promoters, can be used.

[0128]The coding sequence of the fusion protein includes a PDZ domain and
an immobilization domain as described elsewhere herein. Polynucleotides
encoding the amino acid sequence for each domain can be obtained in a
variety of ways known in the art; typically the polynucleotides are
obtained by PCR amplification of cloned plasmids, cDNA libraries, and
cDNA generated by reverse transcription of RNA, using primers designed
based on sequences determined by the practitioner or, more often,
publicly available (e.g., through GenBank). The primers include linker
regions (e.g., sequences including restriction sites) to facilitate
cloning and manipulation in production of the fusion construct. The
polynucleotides corresponding to the PDZ and immobilization regions are
joined in-frame to produce the fusion protein-encoding sequence.

[0129]The fusion proteins of the invention may be expressed as secreted
proteins (e.g., by including the signal sequence encoding DNA in the
fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or as
nonsecreted proteins.

[0130]In some embodiments, the PDZ-containing proteins are immobilized on
a solid surface. The substrate to which the polypeptide is bound may in
any of a variety of forms, e.g., a microtiter dish, a test tube, a
dipstick, a microcentrifuge tube, a bead, a spinnable disk, and the like.
Suitable materials include glass, plastic (e.g., polyethylene, PVC,
polypropylene, polystyrene, and the like), protein, paper, carbohydrate,
lipid monolayer or supported lipid bilayer, and other solid supports.
Other materials that may be employed include ceramics, metals,
metalloids, semiconductive materials, cements and the like.

[0131]In some embodiments, the fusion proteins are organized as an array.
The term "array," as used herein, refers to an ordered arrangement of
immobilized fusion proteins, in which particular different fusion
proteins (i.e., having different PDZ domains) are located at different
predetermined sites on the substrate. Because the location of particular
fusion proteins on the array is known, binding at that location can be
correlated with binding to the PDZ domain situated at that location.
Immobilization of fusion proteins on beads (individually or in groups) is
another particularly useful approach. In one embodiment, individual
fusion proteins are immobilized on beads. In one embodiment, mixtures of
distinguishable beads are used. Distinguishable beads are beads that can
be separated from each other on the basis of a property such as size,
magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead
coated with protein A can be separated from a bead not coated with
protein A by using IgG affinity methods). Binding to particular PDZ
domain may be determined; similarly, the effect of test compounds (I.e.,
agonists and antagonists of binding) may be determined.

[0132]Methods for immobilizing proteins are well known in the art, and
include covalent and non-covalent methods.

[0133]One suitable immobilization method is antibody-mediated
immobilization. According to this method, an antibody specific for the
sequence of an "immobilization domain" of the PDZ-domain containing
protein is itself immobilized on the substrate (e.g., by adsorption). One
advantage of this approach is that a single antibody may be adhered to
the substrate and used for immobilization of a number of polypeptides
(sharing the same immobilization domain). For example, an immobilization
domain consisting of poly-histidine (Bush et al, 1991, J. Biol Chem
266:13811-14) can be bound by an anti-histidine monoclonal antibody (R&D
Systems, Minneapolis, Minn.); an immobilization domain consisting of
secreted alkaline phosphatase ("SEAP") (Berger et al, 1988, Gene 66:
1-10) can be bound by anti-SEAP (Sigma Chemical Company, St. Louis, Mo.);
an immobilization domain consisting of a FLAG epitope can be bound by
anti-FLAG. Other ligand-antiligand immobilization methods are also
suitable (e.g., an immobilization domain consisting of protein A
sequences (Harlow and Lane, 1988, Antibodies A laboratory Manual, Cold
Spring Harbor Laboratory; Sigma Chemical Co., St. Louis, Mo.) can be
bound by IgG; and an immobilization domain consisting of strepavidin can
be bound by biotin (Harlow & Lane, supra; Sigma Chemical Co., St. Louis,
Mo.). In a preferred embodiment, the immobilization domain is a GST
moiety, as described herein.

[0134]When antibody-mediated immobilization methods are used, glass and
plastic are especially useful substrates. The substrates may be printed
with a hydrophobic (e.g., Teflon) mask to form wells. Preprinted glass
slides with 3, 10 and 21 wells per 14.5 cm2 slide "working area" are
available from, e.g., SPI Supplies, West Chester, Pa.; also see U.S. Pat.
No. 4,011,350). In certain applications, a large format (12.4
cm×8.3 cm) glass slide is printed in a 96 well format is used; this
format facilitates the use of automated liquid handling equipment and
utilization of 96 well format plate readers of various types
(fluorescent, colorimetric, scintillation). However, higher densities may
be used (e.g., more than 10 or 100 polypeptides per cm2). See, e.g.,
MacBeath et al, 2000, Science 289:1760-63.

[0135]Typically, antibodies are bound to substrates (e.g., glass
substrates) by adsorption. Suitable adsorption conditions are well known
in the art and include incubation of 0.5-50 μg/ml (e.g., 10 μg/ml)
mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES,
acetate buffers, pHs 6.5 to 8, at 4° C.) to 37° C. and from
1 hr to more than 24 hours.

[0136]Proteins may be covalently bound or noncovalently attached through
nonspecific bonding. If covalent bonding between a the fusion protein and
the surface is desired, the surface will usually be polyfunctional or be
capable of being polyfunctionalized. Functional groups which may be
present on the surface and used for linking can include carboxylic acids,
aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups,
mercapto groups and the like. The manner of linking a wide variety of
compounds to various surfaces is well known and is amply illustrated in
the literature.

[0138]In one aspect, the invention provides an assay in which biotinylated
candidate PL peptides are immobilized on an avidin coated surface. The
binding of PDZ-domain fusion protein to this surface is then measured. In
a preferred embodiment, the PDZ-domain fusion protein is a GST/PDZ fusion
protein and the assay is carried out as follows:

[0139](1) Avidin is bound to a surface, e.g. a protein binding surface. In
one embodiment, avidin is bound to a polystyrene 96 well plate (e.g.,
Nunc Polysorb (cat #475094) by addition of 100 μL per well of 20
μg/mL of avidin (Pierce) in phosphate buffered saline without calcium
and magnesium, pH 7.4 ("PBS", GibcoBRL) at 4° C. for 12 hours. The
plate is then treated to block nonspecific interactions by addition of
200 μL per well of PBS containing 2 g per 100 mL protease-free bovine
serum albumin ("PBS/BSA") for 2 hours at 4° C. The plate is then
washed 3 times with PBS by repeatedly adding 200 μL per well of PBS to
each well of the, plate and then dumping the contents of the plate into a
waste container and tapping the plate gently on a dry surface.

[0140](2) Biotinylated PL peptides (or candidate PL peptides, e.g. see
TABLE 4) are immobilized on the surface of wells of the plate by addition
of 50 μL per well of 0.4 μM peptide in PBS/BSA for 30 minutes at
4° C. Usually, each different peptide is added to at least eight
different wells so that multiple measurements (e.g. duplicates and also
measurements using different (3ST/PDZ-domain fusion proteins and a GST
alone negative control) can be made, and also additional negative control
wells are prepared in which no peptide is immobilized. Following
immobilization of the PL peptide on the surface, the plate is washed 3
times with PBS.

[0141](3) GST/PDZ-domain fusion protein (prepared as described supra) is
allowed to react with the surface by addition of 50 μL per well of a
solution containing 5 μg/mL GST/PDZ-domain fusion protein in PBS/BSA
for 2 hours at 4° C. As a negative control, GST alone (i.e. not a
fusion protein) is added to specified wells, generally at least 2 wells
(i.e. duplicate measurements) for each immobilized peptide. After the 2
hour reaction, the plate is washed 3 times with PBS to remove unbound
fusion protein.

[0142](4) The binding of the GST/PDZ-domain fusion protein to the
avidin-biotinylated peptide surface can be detected using a variety of
methods, and detectors known in the art. In one embodiment, 50 μL per
well of an anti-GST antibody in PBS/BSA (e.g. 2.5 μg/mL of polyclonal
goat-anti-GST antibody, Pierce) is added to the plate and allowed to
react for 20 minutes at 4° C. The plate is washed 3 times with PBS
and a second, delectably labeled antibody is added. In one embodiment, 50
μL per well of 2.5 μg/mL of horseradish peroxidase (HRP)-conjugated
polyclonal rabbit anti-goat immunoglobulin antibody is added to the plate
and allowed to react for 20 minutes at 4° C. The plate is washed 5
times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed by
addition of 100 μL per well of HRP-substrate solution (TMB, Dako) for
20 minutes at room temperature (RT). The reaction of the HRP and its
substrate is terminated by the addition of 100 μL per well of 1 M
sulfuric acid and the optical density (O.D.) of each well of the plate is
read at 450 nm.

[0143](5) Specific binding of a PL peptide and a PDZ-domain polypeptide is
detected by comparing the signal from the well(s) in which the PL peptide
and PDZ domain polypeptide are combined with the background signal(s).
The background signal is the signal found in the negative controls.
Typically a specific or selective reaction will be at least twice
background signal, more typically more than 5 times background, and most
typically 10 or more times the background signal. In addition, a
statistically significant reaction will involve multiple measurements of
the reaction with the signal and the background differing by at least two
standard errors, more typically four standard errors, and most typically
six or more standard errors. Correspondingly, a statistical test (e.g. a
T-test) comparing repeated measurements of the signal with repeated
measurements of the background will result in a p-value <0.05, more
typically a p-value <0.01, and most typically a p-value <0.001 or
less.

[0144]As noted, in an embodiment of the "A" assay, the signal from binding
of a GST/PDZ-domain fusion protein to an avidin surface not exposed to
(i.e. not covered with) the PL peptide is one suitable negative control
(sometimes referred to as "B"). The signal from binding of GST
polypeptide alone (i.e. not a fusion protein) to an avidin-coated surface
that has been exposed to (i.e. covered with) the PL peptide is a second
suitable negative control (sometimes referred to as "B2"). Because all
measurements are done in multiples (i.e. at least duplicate) the
arithmetic mean (or, equivalently, average) of several measurements is
used in determining the binding, and the standard error of the mean is
used in determining the probable error in the measurement of the binding.
The standard error of the mean of N measurements equals the square root
of the following: the sum of the squares of the difference between each
measurement and the mean, divided by the product of (N) and (N-1). Thus,
in one embodiment, specific binding of the PDZ protein to the plate-bound
PL peptide is determined by comparing the mean signal ("mean S") and
standard error of the signal ("SE") for a particular PL-PDZ combination
with the mean B1 and/or mean B2. In TABLE 2, binding was detected to be
specific (denoted by an "A" in the matrix) when (1) the, mean S was at
least twice the mean B1 and at least twice the mean B2 and (2) the mean S
was at least six standard errors (six SE) greater than both the mean B1
and the mean B2. In addition, in the experiments summarized in TABLE 2,
an additional criterion was used to ensure that none of the interactions
defined as specific arose from a combined tendency of both the particular
PDZ fusion protein and PL peptide tested to each give a higher than usual
background. This criteria was that (3) the mean S was at least twenty
times the product of the mean B1 and the mean B2. The factor twenty times
reflects that at least one of B1 and B2 is generally less than 0.1 O.D.
units, and therefore twenty times the product of the mean B1 and the mean
B2 is generally less than twice the mean B1 and twice the mean B2, making
criteria (3) less stringent than criteria (1). Only in a few cases where
the mean B1 and the mean B2 are both greater than 0.1 O.D. units (i.e.
both the particular PDZ fusion protein and PL peptide tested tend to give
a higher than usual background) is criteria (3) more stringent than
criteria (1).

[0146]In one aspect, the invention provides an assay in which a GST/PDZ
fusion protein is immobilized on a surface ("G" assay). The binding of
labeled PL peptide (as listed in TABLE 4) to this surface is then
measured. In a preferred embodiment, the assay is carried out as follows:

[0147](1) A PDZ-domain polypeptide is bound to a surface, e.g. a protein
binding surface. In a preferred embodiment, a GST/PDZ fusion protein
containing one or more PDZ domains is bound to a polystyrene 96-well
plate. The GST/PDZ fusion protein can be bound to the plate by any of a
variety of standard methods known to one of skill in the art, although
some care must be taken that the process of binding the fusion protein to
the plate does not alter the ligand-binding properties of the PDZ domain.
In one embodiment, the GST/PDZ fusion protein is bound via an anti-GST
antibody that is coated onto the 96-well plate. Adequate binding to the
plate can be achieved when: [0148]a. 100 μL per well of 5 μg/mL
goat anti-GST polyclonal antibody (Pierce) in PBS is added to a
polystyrene 96-well plate (e.g., Nunc Polysorb) at 4° C. for 12
hours. [0149]b. The plate is blocked by addition of 200 μL per well of
PBS/BSA for 2 hours at 4° C. [0150]c. The plate is washed 3 times
with PBS. [0151]d. 50 μL per well of 5 μg/mL GST/PDZ fusion
protein) or, as a negative control, GST polypeptide alone (i.e. not a
fusion protein) in PBS/BSA is added to the plate for 2 hours at 4°
C. [0152]e. the plate is again washed 3 times with PBS.

[0153](2) Biotinylated PL peptides (or candidate PL peptides, e.g. as
shown in TABLE 4) are allowed to react with the surface by addition of 50
μL per well of 20 μM solution of the biotinylated peptide in
PBS/BSA for 10 minutes at 4° C., followed by an additional 20
minute incubation at 25° C. The plate is washed 3 times with ice
cold PBS.

[0154](3) The binding of the biotinylated peptide to the GST/PDZ fusion
protein surface can be detected using a variety of methods and detectors
known to one of skill in the art. In one embodiment, 100 μL per well
of 0.5 μg/mL streptavidin-horse radish peroxidase (HRP) conjugate
dissolved in BSA/PBS is added and allowed to react for 20 minutes at
4° C. The plate is then washed 5 times with 50 mM Tris pH 8.0
containing 0.2% Tween 20, and developed by addition of 100 μL per well
of HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature
(RT). The reaction of the HRP and its substrate is terminated by addition
of 100 μL per well of 1 M sulfuric acid, and the optical density
(O.D.) of each well of the plate is read at 450 μm.

[0155](4) Specific binding of a PL peptide and a PDZ domain polypeptide is
determined by comparing the signal from the well(s) in which the PL
peptide and PDZ domain polypeptide are combined, with the background
signal(s). The background signal is the signal found in the negative
control(s). Typically a specific or selective reaction will be at least
twice background signal, more typically more than 5 times background, and
most typically 10 or more times the background signal. In addition, a
statistically significant reaction will involve multiple measurements of
the reaction with the signal and the background differing by at least two
standard errors, more typically four standard errors, and most typically
six or more standard errors. Correspondingly, a statistical test (e.g. a
T-test) comparing repeated measurements of the signal with--repeated
measurements of the background will result in a p-value <0.05, more
typically a p-value <0.01, and most typically a p-value <0.001 or
less. As noted, in an embodiment of the "G" assay, the signal from
binding of a given PL peptide to immobilized (surface bound) GST
polypeptide alone is one suitable negative control (sometimes referred to
as "B1"). Because all measurement are done in multiples (i.e. at least
duplicate) the arithmetic mean (or, equivalently, average.) of several
measurements is used in determining the binding, and the standard error
of the mean is used in determining the probable error in the measurement
of the binding. The standard error of the mean of N measurements equals
the square root of the following: the sum of the squares of the
difference between each measurement and the mean, divided by the product
of (N) and (N-1). Thus, in one embodiment, specific binding of the PDZ
protein to the platebound peptide is determined by comparing the mean
signal ("mean S") and standard error of the signal ("SE") for a
particular PL-PDZ combination with the mean B1. In experiments summarized
in TABLE 2, binding was determined to be specific (denoted by a "G" in
the matrix) when (1) the mean S was at least twice the mean B1 and (2)
the mean S was at least six standard errors (six SE) greater than the
mean B1. Results of exemplary "G" assays are shown in FIGS. 1A-1D.

[0156]6.2.4 Assay Variations

[0157]As discussed supra, it will be appreciated that many of the steps in
the above-described assays can be varied, for example, various substrates
can be used for binding the PL and PDZ-containing proteins; different
types of PDZ containing fusion proteins can be used; different labels for
detecting PDZ/PL interactions can be employed; and different ways of
detection can be used.

[0158]The PDZ-PL detection assays can employ a variety of surfaces to bind
the PL and PDZ-containing proteins. For example, a surface can be an
"assay plate" which is formed from a material (e.g. polystyrene) which
optimizes adherence of either the PL protein or PDZ-containing protein
thereto. Generally, the individual wells of the assay plate will have a
high surface area to volume ratio and therefore a suitable shape is a
flat bottom well (where the proteins of the assays are adherent). Other
surfaces include, but are not limited to, polystyrene or glass beads,
polystyrene or glass slides, and alike.

[0159]For example, the assay plate can be a "microtiter" plate. The term
"microtiter" plate when used herein refers to a multiwell assay plate,
e.g., having between about 30 to 200 individual wells, usually 96 wells.
Alternatively, high density arrays can be used. Often, the individual
wells of the microtiter plate will hold a maximum volume of about 250
μl. Conveniently, the assay plate is a 96 well polystyrene plate (such
as that sold by Becton Dickinson Labware, Lincoln Park, N.J.), which
allows for automation and high throughput screening. Other surfaces
include polystyrene microtiter ELISA plates such as that sold by Nunc
Maxisorp, Inter Med, Denmark. Often, about 50 μl to 300 μl, more
preferably 100 μl to 200 μl, of an aqueous sample comprising
buffers suspended therein will be added to each well of the assay plate.

[0160]The detectable labels of the invention can be any detectable
compound or composition which is conjugated directly or indirectly with a
molecule (such as described above). The label can be detectable by itself
(e.g., radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, can catalyze a chemical alteration of a substrate
compound or composition which is detectable. The preferred label is an
enzymatic one which catalyzes a color change of a non-radioactive color
reagent.

[0161]Sometimes, the label is indirectly conjugated with the antibody. One
of skill is aware of various techniques for indirect conjugation. For
example, the antibody can be conjugated with biotin and any of the
categories of labels mentioned above can be conjugated with avidin, or
vice versa (see also "A" and "G" assay above). Biotin binds selectively
to avidin and thus, the label can be conjugated with the antibody in this
indirect manner. See, Ausubel, supra, for a review of techniques
involving biotin-avidin conjugation and similar assays. Alternatively, to
achieve indirect conjugation of the label with the antibody, the antibody
is conjugated with a small hapten (e.g. digoxin) and one of the different
types of labels mentioned above is conjugated with an anti-hapten
antibody (e.g. anti-digoxin antibody). Thus, indirect conjugation of the
label with the antibody can be achieved.

[0162]Assay variations can include different washing steps. By "washing"
is meant exposing the solid phase to an aqueous solution (usually a
buffer or cell culture media) in such a way that unbound material (e.g.,
non-adhering cells, non-adhering capture agent, unbound ligand, receptor,
receptor construct, cell lysate, or HRP antibody) is removed therefrom.
To reduce background noise, it is convenient to include a detergent
(e.g., Triton X) in the washing solution. Usually, the aqueous washing
solution is decanted from the wells of the assay plate following washing.
Conveniently, washing can be achieved using an automated washing device.
Sometimes, several washing steps (e.g., between about 1 to 10 washing
steps) can be required.

[0163]Various buffers can also be used in PDZ-PL detection assays. For
example, various blocking buffers can be used to reduce assay background.
The term "blocking buffer" refers to an aqueous, pH buffered solution
containing at least one blocking compound which is able to bind to
exposed surfaces of the substrate which are not coated with a PL or
PDZ-containing protein. The blocking compound is normally a protein such
as bovine serum albumin (BSA), gelatin, casein or milk powder and does
not cross-react with any of the reagents in the assay. The block buffer
is generally provided at a pH between about 7 to 7.5 and suitable
buffering agents include phosphate and TRIS.

[0164]Various enzyme-substrate combinations can also be utilized in
detecting PDZ-PL interactions. Examples of enzyme-substrate combinations
include, for example:

[0165](i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a
substrate,

[0169]Numerous other enzyme-substrate combinations are available to those
skilled in the art. For a general review of these, see U.S. Pat. Nos.
4,275,149 and 4,318,980, both of which are herein incorporated by
reference.

[0170]Further, it will be appreciated that, although, for convenience, the
present discussion primarily refers antagonists of PDZ-PL interactions,
agonists of PDZ-PL interactions can be identified using the methods
disclosed herein or readily apparent variations thereof.

[0171]6.2.5 Results of PDL-PL Interaction Assays

[0172]TABLE 2, supra, shows the results of assays in which specific
binding was detected using the "A" and "G" assays described herein. The
top row of the table specifies the source of the PDZ domain used in the
GST-PDZ fusion proteins (see TABLE 3). The first column lists the cell
surface proteins from which C-terminal peptide sequences were derived and
the second column ("code") identifies the peptide used in the assay (see
TABLE 4). The third column, "Seq" provides the sequence of the four (4)
C-terminal residues of the cell surface protein and peptide. In the
matrix, "A" indicates specific binding as detected in the "A" assay. "G"
indicates specific binding as shown in the "G" assay. A blank indicates
that no specific binding was detected using the "A" or "G" assays. An
asterisk (*) indicates that a pairwise interaction between the PDZ
protein and the cell surface protein (or subdomains of either) has been
described by others.

[0173]6.2.5.1 New PL Motifs

[0174]As noted supra, TABLE 2 shows the results of assays (referred to as
"PRISM MATRIX") to detect binding between PDZ proteins and candidate PL
peptides. A number of specific PDZ-PL interactions are identified by the
MATRIX and key amino acids and positions important in PDZ binding ("PL
motifs") are deduced from these results. Not only is the MATRIX useful to
catalog comprehensively PDZ-PL binding combinations, the assay can
further aid in the rapid discovery and characterization of novel PL
proteins and PL motifs to help in rational drug design and synthesis of
PL-PDZ interaction inhibitors.

[0175]Other investigators have reported certain PL motifs important in PDZ
binding, e.g., the C-terminal motifs S/T-X-V/I/L (forDLGl) and
Y/F-Y/F-I/L/F for MPP1 (see, Doyle et al., 1996, Cell 85, 1067; Songyang
et al., 1997, Science 275, 73). However, the reported motifs are not
sufficiently specific (i.e. a large number of proteins meet these
criteria yet are not necessarily actual PDZ ligands) and cover only a
small number of PDZ proteins (approximately 10). The PRISM MATRIX can be
used to determine ligand specificity and to deduce ligand binding motifs
for any PDZ protein because it can precisely determine sequences of amino
acids that do or do not result in specific PDZ binding. In addition, the
assay has revealed a significant of new PDZ domain binding motifs (i.e.
PL motifs): C-terminal sequence of CD6, ISAA (SEQ ID NO:14); C-terminal
sequence of CD49E, TSDA (SEQ ID NO:24); C-terminal sequence of CD49F,
TSDA (SEQ ID NO:24); C-terminal sequence of CLASP-1, SAEV (SEQ ID
NO:289); C-terminal sequence of CLASP-4, YAEV (SEQ ID NO:228); C-terminal
sequence of CD44, KIGV (SEQ ID NO:104); C-terminal sequence of IL5R, DSVF
(SEQ ID NO:94); C-terminal sequence of BLR-1, LTTF (SEQ ID NO:253).
Identification of these novel PL sequences allows the definition of novel
PL motifs (See TABLE 5A, infra). The specificity with which these novel
motifs are defined is enhanced by the fact that the MATRIX reports both
positive results (i.e. PDZ-PL) combinations that result in specific
binding interactions) and negative results (i.e. PDZ-PL combinations that
do not result in specific binding). For example, the C-terminal sequence
of CD6, SAA and the C-terminal sequence of CD49E, SDA bind to the
PDZ-domain polypeptide 41.8 while the related C-terminal sequence of
CD166, TEA and C-terminal sequence of CD148, YIA do not. This identifies
the novel PL motif (Motif 1, infra) of polypeptides terminating in
alanine with serine at the -2 position and excludes polypeptides with
threonine and tyrosine at the -2 position. This motif is therefore more
specific than most previously identified motifs. Other novel motifs are
described in TABLE 5A.

[0177]TABLE 2 is arranged so that both the PDZ/GST fusion proteins and the
PL peptide ligands are ordered with structurally similar molecules close
to each other. In preparing TABLE 2 and carrying out the experiments used
to create this table, the PDZ domains of each of the GST-PDZ fusion
proteins were ranked for amino acid sequence similarity using the CLUSTAL
multiple sequence alignment software package. Proteins with greatest
sequence similarity are closest to each other in the matrix. The PL
peptide ligands were also ordered on the basis of amino acid similarity,
but with weight given to residues reported to be important in PDZ binding
(Doyle et al., 1996, Cell 85, 1067). In particular, peptides were first
ordered based on the most C-terminal residue (zero position) in the
following amino acid order: G, A, C, S, T, N, Q, D, E, H, K, R, V, I, L,
M, P, F, Y, W. Among peptides with identical C-termini, the same ranking
scheme was then applied to the next most important residue for peptide
binding, the -2 position, followed by the -1 position and the -3
position. (In an alternative approach, the GST-PDZ fusions can also be
arranged to give additional weight to residues known to be important for
ligand binding when aligning the proteins.)

[0178]Regions of the TABLE 1 matrix that are densely filled with
significant binding interactions indicate that a particular ligand
structure tends to bind to a particular family of PDZ domains. The
results indicate that PDZ domains with similar structures bind ligands
with similar structures, as indicated by the presence in the matrix of
some clusters of many significant binding interactions and other areas of
few significant binding interactions. These results reveal a number of
structural relationships between different PDZ proteins and their
ligands. The understanding of these structural relationships can be used
to scan databases for probable ligands of specific PDZ proteins,
facilitating the design of inhibitors of such novel interactions and the
prediction of the side-effect profile of such inhibitors.

[0179]The most striking example of a cluster occurs in columns 1-5 of the
matrix (PDZ domains from CASK, MPP1, DLG1, PSD95, and NeDLG). In our
assays, CASK bound significantly to only one of the tested ligands,
neurexin (this interaction was previously identified in neuronal cells).
MPP1, the closest relative to CASK in the ordering system used, bound a
set of five different ligands, including neurexin. All of the MPP1
ligands identified are structurally similar in that they terminate in
valine (V) and have a charged amino acid (E or K) at the -3 position from
the C-terminus ((C-terminal motif: E/K-X-X-V)

[0180]Out of the five MPP1 ligands identified, four of these also bind to
DLG1, the next most similar protein to CASK and MPP1. DLG1 is closely
related to two other proteins, PSD95 and NeDLG. Each of these three
proteins binds ligands that terminate in the motif S/T/Y--X-V/I/L, as
previously described (Songyang et al., 1997, Science 275, 73).
Interestingly, if the terminal ligand residue is valine, other previously
unidentified residues at the -2 position are compatible with binding,
with the C-terminal sequences AEV and ELV resulting in significant
binding events. While this reflects a previously unrecognized flexibility
in PDZ-ligand binding, the specificity of the interactions detected in
our assays is reflected in the large areas at the top and bottom of
columns 1-5 that contain no significant binding event. These areas
reflect that none of the potential ligands terminating in residues other
than V/mL bound significantly to CASK, MPP1, DLG1, PSD95, or NeDLG. (With
respect to MPP1, it worth noting that Glycophorin C, a known ligand of
MPP1, Marfatia et al., 1997, J. Biol. Chem. 272, 24191, did not bind
significantly to MPP1 in our tests, but does terminate in isoleucine, an
amino acids very similar to valine, and does have a charged residue at
the -3 position of the C-terminus.)

[0181]Other smaller clusters on the matrix also provide valuable
information about the ligand specificity of certain PDZ domains. For
example, five ligands bind significantly in both the "G" and the "A"
assays to 41.8 kD protein, a previously unstudied PDZ domain-containing
protein.

[0182]All of these ligands terminate in A, V, I, or L, and four of five
have serine (S) at the -2 position, defining the C-terminal ligand motif
S-X-A/V/mL for this PDZ domain. Similarly, two of three ligands that bind
specifically to KIAAO561 fit the C-terminal motif X* -S/T-D/E-V/mL where
X* is any non-aromatic amino acid. Interestingly, one of these ligands
also binds to KIAA 0561's closest relative, TAX2. Likewise, the two
ligands that bind to prIL16 both have a charged amino acid at the -3
position, a hydrophobic amino acid at the -2 position, and valine at the
terminal position. A final interesting example of defining the ligand
specificity of a PDZ domain involves PTN-4. Although the two ligands that
bind significantly to PTN-4 are not closely related at their C-terminal 2
residues, both fit the C-terminal motif D/E-S-X-V/I/L/F/Y. These motifs
characterizing the ligand specificity of particular PDZ proteins are
summarized in TABLE 5B. Such motifs allow searches of databases for novel
ligands (PL proteins) binding to these particular PDZ proteins. Knowledge
of the PL proteins that bind to these PDZ proteins allows the design of
inhibitors of these interactions, using the methods described herein,
infra. In addition, knowledge of the set of PL proteins that bind to a
particular PDZ protein can be used to predict the utility and the side
effects of compounds that target this PDZ protein.

[0183]The "A" and "G" assays of the invention can be used to determine the
"apparent affinity" of binding of a PDZ ligand peptide to a PDZ-domain
polypeptide. Apparent affinity is determined based on the concentration
of one molecule required to saturate the binding of a second molecule
(e.g., the binding of a ligand to a receptor). Two particularly useful
approaches for quantitation of apparent affinity of PDZ-ligand binding
are provided infra.

[0184](1) A GST/PDZ fusion protein, as well as GST alone as a negative
control, are bound to a surface (e.g., a 96-well plate) and the surface
blocked and washed as described supra for the "G" assay.

[0185](2) 50 μL per well of a solution of biotinylated PL peptide (e.g.
as shown in TABLE 4) is added to the surface in increasing concentrations
in PBS/BSA (e.g. at 0.1 μM, 0.33 μM, 1 μM, 3.3 μM, 10 μM,
33 μM, and 100 μM). In one embodiment, the PL peptide is allowed to
react with the bound GST/PDZ fusion protein (as well as the GST alone
negative control) for 10 minutes at 4 C followed by 20 minutes at 25 C.
The plate is washed 3 times with ice cold PBS to remove unbound labeled
peptide.

[0186](3) The binding of the PL peptide to the immobilized PDZ-domain
polypeptide is detected as described supra for the "G" assay.

[0187](4) For each concentration of peptide, the net binding signal is
determined by subtracting the binding of the peptide to GST alone from
the binding of the peptide to the GST/PDZ fusion protein. The net binding
signal is then plotted as a function of ligand concentration and the plot
is fit (e.g. by using the Kaleidagraph software package curve fitting
algorithm) to the following equation, where "Signal.sub.[ligand]" is the
net binding signal at PL peptide concentration "[ligand]," "Kd" is the
apparent affinity of the binding event, and "Saturation Binding" is a
constant determined by the curve fitting algorithm to optimize the fit to
the experimental data:

Signal.sub.[ligand]=Saturation Binding×([ligand]/([ligand]+Kd))

[0188]For reliable application of the above equation it is necessary that
the highest peptide ligand concentration successfully tested
experimentally be greater than, or at least similar to, the calculated Kd
(equivalently, the maximum observed binding should be similar to the
calculated saturation binding). In cases where satisfying the above
criteria proves difficult, an alternative approach (infra) can be used.

[0190](1) A fixed concentration of a PDZ-domain polypeptide and increasing
concentrations of a labeled PL peptide (labeled with, for example, biotin
or fluorescein, see TABLE 4 for representative peptide amino acid
sequences) are mixed together in solution and allowed to react. In one
embodiment, preferred peptide concentrations are 0.1 μM, 1 μM, 10
μM, 100 μM, 1 mM. In various embodiments, appropriate reaction
times can range from 10 minutes to 2 days at temperatures ranging from 4
C to 37 C. In some embodiments, the identical reaction can also be
carried out using a non-PDZ domain-containing protein as a control (e.g.,
if the PDZ-domain polypeptide is fusion protein, the fusion partner can
be used).

[0191](2) PDZ-ligand complexes can be separated from unbound labeled
peptide using a variety of methods known in the art. For example, the
complexes can be separated using high performance size-exclusion
chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity
9:699), affinity chromatography (e.g., using glutathione sepharose
beads), and affinity absorption (e.g., by binding to an anti-GST-coated
plate as described supra).

[0192](3) The PDZ-ligand complex is detected based on presence of the
label on the peptide ligand using a variety of methods and detectors
known to one of skill in the art. For example, if the label is
fluorescein and the separation is achieved using HPSEC, an in-line
fluorescence detector can be used. The binding can also be detected as
described supra for the G assay.

[0193](4) The PDZ-ligand binding signal is plotted as a function of ligand
concentration and the plot is fit. (e.g., by using the Kaleidagraph
software package curve fitting algorithm) to the following equation,
where "Signal.sub.[ligand]" is the binding signal at PL peptide
concentration "[ligand]," "Kd" is the apparent affinity of the binding
event, and "Saturation Binding" is a constant determined by the curve
fitting algorithm to optimize the fit to the experimental data:

Signal.sub.[Ligand]=Saturation Binding×([ligand]/([ligand+Kd])

[0194]Measurement of the affinity of a labeled peptide ligand binding to a
PDZ-domain polypeptide n is useful because knowledge of the affinity (or
apparent affinity) of this interaction allows rational design of
inhibitors of the interaction with known potency (See EXAMPLE 2). The
potency of inhibitors in inhibition would be similar to (i.e. within
one-order of magnitude of) the apparent affinity of the labeled peptide
ligand binding to the PDZ-domain.

[0195]Thus, in one aspect, the invention provides a method of determining
the apparent affinity of binding between a PDZ domain and a ligand by
immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain
on a surface, contacting the immobilized polypeptide with a plurality of
different concentrations of the ligand, determining the amount of binding
of the ligand to the immobilized polypeptide at each of the
concentrations of ligand, and calculating the apparent affinity of the
binding based on that data. Typically, the polypeptide comprising the PDZ
domain and a non-PDZ domain is a fusion protein. In one embodiment, the
e.g., fusion protein is GST-PDZ fusion protein, but other polypeptides
can also be used (e.g., a fusion protein including a PDZ domain and any
of a variety of epitope tags, biotinylation signals and the like) so long
as the polypeptide can be immobilized in an orientation that does not
abolish the ligand binding properties of the PDZ domain, e.g, by
tethering the polypeptide to the surface via the non-PDZ domain via an
anti-domain antibody and leaving the PDZ domain as the free end. It was
discovered, for example, reacting a PDZ-GST fusion polypeptide directly
to a plastic plate provided suboptimal results. The calculation of
binding affinity itself can be determined using any suitable equation
(e.g., as shown supra; also see Cantor and Schimmel (1980) BIOPHYSICAL
CHEMISTRY WH Freeman & Co., San Francisco) or software.

[0196]Thus, in a preferred embodiment, the polypeptide is immobilized by
binding the polypeptide to an immobilized immunoglobulin that binds the
non-PDZ domain (e.g., an anti-GST antibody when a GST-PDZ fusion
polypeptide is used). In a preferred embodiment, the step of contacting
the ligand and PDZ-domain polypeptide is carried out under the conditions
provided supra in the description of the "G" assay. It will be
appreciated that binding assays are conveniently carried out in multiwell
plates (e.g., 24-well, 96-well plates, or 384 well plates).

[0197]The present method has considerable advantages over other methods
for measuring binding affinities PDZ-PL affinities, which typically
involve contacting varying concentrations of a GST-PDZ fusion protein to
a ligand-coated surface. For example, some previously described methods
for determining affinity (e.g., using immobilized ligand and GST-PDZ
protein in solution) did not account for oligomerization state of the
fusion proteins used, resulting in potential errors of more than an order
of magnitude.

[0199]In a specific embodiment, screening can be carried out by contacting
the library members with a hematopoietic cell PDZ-domain polypeptide
immobilized on a solid support (e.g. as described supra in the "G" assay)
and harvesting those library members that bind to the protein. Examples
of such screening methods, termed "panning" techniques are described by
way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et
al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and
in references cited hereinabove.

[0200]In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246;
Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used
to identify molecules that specifically bind to a PDZ domain-containing
protein. Furthermore, the identified molecules are further tested for
their ability to inhibit transmembrane receptor interactions with a PDZ
domain.

[0201]In one aspect of the invention, antagonists of an interaction
between a PDZ protein and a PL protein are identified. In one embodiment,
a modification of the "A" assay described supra is used to identify
antagonists. In one embodiment, a modification of the "G" assay described
supra is used to identify antagonists.

[0202]In one embodiment, screening assays are used to detect molecules
that specifically bind to PDZ domains in hematopoietic cells. Such
molecules are useful as agonists or antagonists of PDZ-protein-mediated
cell function (e.g., cell activation, e.g., T cell activation, vesicle
transport, cytokine release, growth factors, transcriptional changes,
cytoskeletin rearrangement, cell movement, chemotaxis, and the like). In
one embodiment, such assays are performed to screen for leukocyte
activation inhibitors for drug development. The invention thus provides
assays to detect molecules that specifically bind to PDZ
domain-containing proteins in hematopoietic cells. For example,
recombinant cells expressing PDZ domain-encoding nucleic acids can be
used to produce PDZ domains in these assays and to screen for molecules
that bind to the domains. Molecules are contacted with the PDZ domain (or
fragment thereof) under conditions conducive to binding, and then
molecules that specifically bind to such domains are identified. Methods
that can be used to carry out the foregoing are commonly known in the
art.

[0203]It will be appreciated by the ordinarily skilled practitioner that,
in one embodiment, antagonists are identified by conducting the A or G
assays in the presence and absence of a known or candidate antagonist.
When decreased binding is observed in the presence of a compound, that
compound is identified as an antagonist. Increased binding in the
presence of a compound signifies that the compound is an agonist.

[0204]For example, in one assay, a test compound can be identified as an
inhibitor (antagonist) of binding between a PDZ protein and a PL protein
by contacting a PDZ domain polypeptide and a PL peptide in the presence
and absence of the test compound, under conditions in which they would
(but for the presence of the test compound) form a complex, and detecting
the formation of the complex in the presence and absence of the test
compound. It will be appreciated that less complex formation in the
presence of the test compound than in the absence of the compound
indicates that the test compound is an inhibitor of a PDZ protein --PL
protein binding. In various embodiments, the PL peptide comprises an
amino acid sequence substantially identical to the C-terminal sequence of
a PL protein (e.g., CD6, CD49E, CD49F, CD138, CLASP-1, CLASP-4, VCAM1,
CLASP-2, CD95, DNAM-1, CD83, CD44, CD4, CD97, Neurexin, CD3n, DOCK2,
CD34, FceRIb, or FasLigand).

[0205]In one embodiment, the "G" assay is used in the presence or absence
of an candidate inhibitor. In one embodiment, the "A" assay is used in
the presence or absence of a candidate inhibitor.

[0206]In one embodiment (in which a G assay is used), one or more PDZ
domain-containing GST-fusion proteins are bound to the surface of wells
of a 96-well plate as described supra (with appropriate controls
including nonfusion GST protein). All fusion proteins are bound in
multiple wells so that appropriate controls and statistical analysis can
be done. A test compound in BSA/PBS (typically at multiple different
concentrations) is added to wells. Immediately thereafter, 30 μL of a
detectably labeled (e.g., biotinylated) peptide known to bind to the
relevant PDZ domain (see, e.g., TABLE 2) is added in each of the wells at
a final concentration of, e.g., between about 2 μM and about 40 μM,
typically 5 μM, 15 μM, or 25 μM. This mixture is then allowed to
react with the PDZ fusion protein bound to the surface for 10 minutes at
4° C. followed by 20 minutes at 25° C. The surface is
washed free of unbound peptide three times with ice cold PBS and the
amount of binding of the peptide in the presence and absence of the test
compound is determined. Usually, the level of binding is measured for
each set of replica wells (e.g. duplicates) by subtracting the mean GST
alone background from the mean of the raw measurement of peptide binding
in these wells.

[0207]In an alternative embodiment, the A assay is carried out in the
presence or absence of an test candidate to identify inhibitors of PL-PDZ
interactions.

[0208]In one embodiment, a test compound is determined to be a specific
inhibitor of the binding of the PDZ domain (P) and a PL (L) sequence
when, at a test compound concentration of less than or equal to 1 mM
(e.g., less than or equal to: 500 μM, 100 μM, 10 μM, 1 μM,
100 nM or 1 nM) the binding of P to L in the presence of the test
compound less than about 50% of the binding in the absence of the test
compound. (in various embodiments, less than about 25%, less than about
10%, or less than about 1%). Preferably, the net signal of binding of P
to L in the presence of the test compound plus six (6) times the standard
error of the signal in the presence of the test compound is less than the
binding signal in the absence of the test compound.

[0209]In one embodiment, assays for an inhibitor are carried out using a
single PDZ protein-PL protein pair (e.g., a PDZ domain fusion protein and
a PL peptide). In a related embodiment, the assays are carried out using
a plurality of pairs, such as a plurality of different pairs listed in
TABLE 2.

[0210]In some embodiments, it is desirable to identify compounds that, at
a given concentration, inhibit the binding of one PL-PDZ pair, but do not
inhibit (or inhibit to a lesser degree) the binding of a specified second
PL-PDZ pair. These antagonists can be identified by carrying out a series
of assays using a candidate inhibitor and different PL-PDZ pairs (e.g.,
as shown in the matrix of TABLE 2) and comparing the results of the
assays. All such pairwise combinations are contemplated by the invention
(e.g., test compound inhibits binding of PL1 to PDZ1 to a
greater degree than it inhibits binding of PL1 to PDZ2 or
PL2 to PDZ2). Importantly, it will be appreciated that, based
on the data provided in TABLE 2 and disclosed herein (and additional data
that can be generated using the methods described herein) inhibitors with
different specificities can readily be designed.

[0211]For example, according to the invention, the Ki ("potency") of an
inhibitor of a PDZ-PL interaction can be determined. Ki is a measure of
the concentration of an inhibitor required to have a biological effect.
For example, administration of an inhibitor of a PDZ-PL interaction in an
amount sufficient to result in an intracellular inhibitor concentration
of at least between about 1 and about 100 Ki is expected to inhibit the
biological response mediated by the target PDZ-PL interaction. In one
aspect of the invention, the Kd measurement of PDZ-PL binding as
determined using the methods supra is used in determining Ki.

[0212]Thus, in one aspect, the invention provides a method of determining
the potency (Ki) of an inhibitor or suspected inhibitor of binding
between a PDZ domain and a ligand by immobilizing a polypeptide
comprising the PDZ domain and a non-PDZ domain on a surface, contacting
the immobilized polypeptide with a plurality of different mixtures of the
ligand and inhibitor, wherein the different mixtures comprise a fixed
amount of ligand and different concentrations of the inhibitor,
determining the amount of ligand bound at the different concentrations of
inhibitor, and calculating the Ki of the binding based on the amount of
ligand bound in the presence of different concentrations of the
inhibitor. In an embodiment, the polypeptide is immobilized by binding
the polypeptide to an immobilized immunoglobulin that binds the non-PDZ
domain. This method, which is based on the "G" assay described supra, is
particularly suited for high-throughput analysis of the Ki for inhibitors
of PDZ-ligand interactions. Further, using this method, the inhibition of
the PDZ-ligand interaction itself is measured, without distortion of
measurements by avidity effects.

[0213]Typically, at least a portion of the ligand is detectably labeled to
permit easy quantitation of ligand binding.

[0214]It will be appreciated that the concentration of ligand and
concentrations of inhibitor are selected to allow meaningful detection of
inhibition. Thus, the concentration of the ligand whose binding is to be
blocked is close to or less than its binding affinity (e.g., preferably
less than the 5×Kd of the interaction, more preferably less than
2×Kd, most preferably less than 1×Kd). Thus, the ligand is
typically present at a concentration of less than 2 Kd (e.g., between
about 0.01 Kd and about 2 Kd) and the concentrations of the test
inhibitor typically range from 1 nM to 100 μM (e.g. a 4-fold dilution
series with highest concentration 10 μM or 1 mM). In a preferred
embodiment, the Kd is determined using the assay disclosed supra.

[0215]The Ki of the binding can be calculated by any of a variety of
methods routinely used in the art, based on the amount of ligand bound in
the presence of different concentrations of the inhibitor. in an
illustrative embodiment, for example, a plot of labeled ligand binding
versus inhibitor concentration is fit to the equation:

Sinhibitor=S0*Ki/([I]+Ki)

where Sinhibitor is the signal of labeled ligand binding to
immobilized PDZ domain in the presence of inhibitor at concentration [I]
and S0 is the signal in the absence of inhibitor (i.e., [I]=0).
Typically [I] is expressed as a molar concentration.

[0216]In another aspect of the invention, an enhancer (sometimes referred
to as, augmentor or agonist) of binding between a PDZ domain and a ligand
is identified by immobilizing a polypeptide comprising the PDZ domain and
a non-PDZ domain on a surface, contacting the immobilized polypeptide
with the ligand in the presence of a test agent and determining the
amount of ligand bound, and comparing the amount of ligand bound in the
presence of the test agent with the amount of ligand bound by the
polypeptide in the absence of the test agent. At least two-fold (often at
least 5-fold) greater binding in the presence of the test agent compared
to the absence of the test agent indicates that the test agent is an
agent that enhances the binding of the PDZ domain to the ligand. As noted
supra, agents that enhance PDZ-ligand interactions are useful for
disruption (dysregulation) of biological events requiring normal
PDZ-ligand function (e.g., cancer cell division and metastasis, and
activation and migration of immune cells).

[0217]The invention also provides methods for determining the "potency" or
"Kenhancer" of an enhancer of a PDZ-ligand interaction. For example,
according to the invention, the Kenhancer of an enhancer of a PDZ-PL
interaction can be determined, e.g., using the Kd of PDZ-PL binding as
determined using the methods described supra. Kenhancer is a measure
of the concentration of an enhancer expected to have a biological effect.
For example, administration of an enhancer of a PDZ-PL interaction in an
amount sufficient to result in an intracellular inhibitor concentration
of at least between about 0.1 and about 100 Kenhancer (e.g., between
about 0.5 and about 50 Kenhancer) is expected to disrupt the
biological response mediated by the target PDZ-PL interaction.

[0218]Thus, in one aspect the invention provides a method of determining
the potency (Kenhancer) of an enhancer or suspected enhancer of
binding between a PDZ domain and a ligand by immobilizing a polypeptide
comprising the PDZ domain and a non-PDZ domain on a surface, contacting
the immobilized polypeptide with a plurality of different mixtures of the
ligand and enhancer, wherein the different mixtures comprise a fixed
amount of ligand, at least a portion of which is detectably labeled, and
different concentrations of the enhancer, determining the amount of
ligand bound at the different concentrations of enhancer, and calculating
the potency (Kenhancer) of the enhancer from the binding based on
the amount of ligand bound in the presence of different concentrations of
the enhancer. Typically, at least a portion of the ligand is detectably
labeled to permit easy quantitation of ligand binding. This method, which
is based on the "G" assay described supra, is particularly suited for
high-throughput analysis of the Kenhancer for enhancers of
PDZ-ligand interactions.

[0219]It will be appreciated that the concentration of ligand and
concentrations of enhancer are selected to allow meaningful detection of
enhanced binding. Thus, the ligand is typically present at a
concentration of between about 0.01 Kd and about 0.5 Kd and the
concentrations of the test agent/enhancer typically range from 1 nM to 1
mM (e.g. a 4-fold dilution series with highest concentration 10 μM or
1 mM). In a preferred embodiment, the Kd is determined using the assay
disclosed supra.

[0220]The potency of the binding can be determined by a variety of
standard methods based on the amount of ligand bound in the presence of
different concentrations of the enhancer or augmentor. For example, a
plot of labeled ligand binding versus enhancer concentration can be fit
to the equation:

S([E])=S(0)+(S(0)*(Denhancer-1)*[E]/([E]+Kenhancer)

where "Kenhancer" is the potency of the augmenting compound, and
"Denhancer" is the fold-increase in binding of the labeled ligand
obtained with addition of saturating amounts of the enhancing compound,
[E] is the concentration of the enhancer. It will be understood that
saturating amounts are the amount of enhancer such that further addition
does not significantly increase the binding signal. Knowledge of
"Kenhancer" is useful because it describes a concentration of the
augmenting compound in a target cell that will result in a biological
effect due to dysregulation of the PDZ-PL interaction. Typical
therapeutic concentrations are between about 0.1 and about 100
Kenhancer.

Global Analysis of PDZ-PL Interactions

[0221]As described supra, the present invention provides powerful methods
for analysis of PDZ-ligand interactions, including high-throughput
methods such as the "G" assay and affinity assays described supra. In one
embodiment of the invention, the affinity is determined for a particular
ligand and a plurality of PDZ proteins. Typically the plurality is at
least 5, and often at least 25, or at least 40 different PDZ proteins. In
a preferred embodiment, the plurality of different PDZ proteins are from
a particular tissue (e.g., central nervous system, spleen, cardiac
muscle, kidney) or a particular class or type of cell, (e.g., a
hematopoietic cell, a lymphocyte, a neuron) and the like. In a most
preferred embodiment, the plurality of different PDZ proteins represents
a substantial fraction (e.g., typically a majority, more often at least
80%) of all of the PDZ proteins known to be, or suspected of being,
expressed in the tissue or cell(s), e.g., all of the PDZ proteins known
to be present in lymphocytes. In an embodiment, the plurality is at least
50%, usually at least 80%, at least 90% or all of the PDZ proteins
disclosed herein as being expressed in hematopoietic cells (see Table 6).

[0222]In one embodiment of the invention, the binding of a ligand to the
plurality of PDZ proteins is determined. Using this method, it is
possible to identify a particular PDZ domain bound with particular
specificity by the ligand. The binding may be designated as "specific" if
the affinity of the ligand to the particular PDZ domain is at least
2-fold that of the binding to other PDZ domains in the plurality (e.g.,
present in that cell type). The binding is deemed "very specific" if the
affinity is at least 10-fold higher than to any other PDZ in the
plurality or, alternatively, at least 10-fold higher than to at least
90%, more often 95% of the other PDZs in a defined plurality. Similarly,
the binding is deemed "exceedingly specific" if it is at least 100-fold
higher. For example, a ligand could bind to 2 different PDZs with an
affinity of 1 μM and to no other PDZs out of a set 40 with an affinity
of less than 100 μM. This would constitute specific binding to those 2
PDZs. Similar measures of specificity are used to describe binding of a
PDZ to a plurality of PLs.

[0223]It will be recognized that high specificity PDZ-PL interactions
represent potentially more valuable targets for achieving a desired
biological effect. The ability of an inhibitor or enhancer to act with
high specificity is often desirable. In particular, the most specific
PDZ-ligand interactions are also the best therapeutic targets, allowing
specific inhibition of the interaction.

[0224]In an embodiment an interaction between a PDZ and a PL is deemed

[0225]Thus, in one embodiment, the invention provides a method of
identifying a high specificity interaction between a particular PDZ
domain and a ligand known or suspected of binding at least one PDZ
domain, by providing a plurality of different immobilized polypeptides,
each of said polypeptides comprising a PDZ domain and a non-PDZ domain;
determining the affinity of the ligand for each of said polypeptides, and
comparing the affinity of binding of the ligand to each of said
polypeptides, wherein an interaction between the ligand and a particular
PDZ domain is deemed to have high specificity when the ligand binds an
immobilized polypeptide comprising the particular PDZ domain with at
least 2-fold higher affinity than to immobilized polypeptides not
comprising the particular PDZ domain.

[0226]In a related aspect, the affinity of binding of a specific PDZ
domain to a plurality of ligands (or suspected ligands) is determined.
For example, in one embodiment, the invention provides a method of
identifying a high specificity interaction between a PDZ domain and a
particular ligand known or suspected of binding at least one PDZ domain,
by providing an immobilized polypeptide comprising the PDZ domain and a
non-PDZ domain; determining the affinity of each of a plurality of
ligands for the polypeptide, and comparing the affinity of binding of
each of the ligands to the polypeptide, wherein an interaction between a
particular ligand and the PDZ domain is deemed to have high specificity
when the ligand binds an immobilized polypeptide comprising the PDZ
domain with at least 2-fold higher affinity than other ligands tested.
Thus, the binding may be designated as "specific" if the affinity of the
PDZ to the particular PL is at least 2-fold that of the binding to other
PLs in the plurality (e.g., present in that cell type). The binding is
deemed "very specific" if the affinity is at least 10-fold higher than to
any other PL in the plurality or, alternatively, at least 10-fold higher
than to at least 90%, more often 95% of the other PLs in a defined
plurality. Similarly, the binding is deemed "exceedingly specific" if it
is at least 100-fold higher. Typically the plurality is at least 5
different ligands, more often at lease 10.

[0227]One discovery of the present inventors relates to the important and
extensive roles played by interactions between PDZ proteins and PL
proteins, particularly in the biological function of hematopoietic cells
and other cells involved in the immune response. Further, it has been
discovered that valuable information can be ascertained by analysis
(e.g., simultaneous analysis) of a large number of PDZ-PL interactions.
In a most preferred embodiment, the analysis encompasses all of the PDZ
proteins expressed in a particular tissue (e.g., spleen) or type or class
of cell (e.g., hematopoietic cell, neuron, lymphocyte, B cell, T cell and
the like). Alternatively, the analysis encompasses at least about 5, or
at least about 10, or at least about 12, or at least about 15 and often
at least 50 different polypeptides; or a substantial fraction (e.g.,
typically a majority, more often at least 80%) of all of the PDZ proteins
known to be, or suspected of being, expressed in the tissue or cell(s),
e.g., all of the PDZ proteins known to be present in lymphocytes. In an
embodiment, the plurality is at least 50%, usually at least 80%, at least
90% or all of the PDZ proteins disclosed herein as being expressed in
hematopoietic cells (see Table 6).

[0229]It will be apparent from this disclosure that analysis of the
relatively large number of different interactions preferably takes place
simultaneously. In this context, "simultaneously" means that the analysis
of several different PDZ-PL interactions (or the effect of a test agent
on such interactions) is assessed means together (e.g., the same day or
same hour). Typically the analysis is carried out in a high throughput
(e.g., robotic) fashion. One advantage of this method of simultaneous
analysis is that it permits rigorous comparison of multiple different
PDZ-PL interactions. For example, as explained in detail elsewhere
herein, simultaneous analysis (and use of the arrays described infra)
facilitates, for example, the direct comparison of the effect of an agent
(e.g., an potential interaction inhibitor) on the interactions between a
substantial portion of PDZs and/or PLs in a tissue or cell.

[0230]Accordingly, in one aspect, the invention provides an array of
immobilized polypeptide comprising the PDZ domain and a non-PDZ domain on
a surface. Typically, the array comprises at least about 5, or at least
about 10, or at least about 12, or at least about 15 and often at least
50 different polypeptides. In one preferred embodiment, the different PDZ
proteins are from a particular tissue (e.g., central nervous system,
spleen, cardiac muscle, kidney) or a particular class or type of cell,
(e.g., a hematopoietic cell, a lymphocyte, a neuron) and the like. In a
most preferred embodiment, the plurality of different PDZ proteins
represents a substantial fraction (e.g., typically a majority, more often
at least 80%) of all of the PDZ proteins known to be, or suspected of
being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins
known to be present in lymphocytes. In an embodiment, the plurality is at
least 50%, usually at least 80%, at least 90% or all of the PDZ proteins
disclosed herein as being expressed in hematopoietic cells (see Table 6).
all of the PDZ proteins known to be present in lymphocytes. In an
embodiment, the plurality is at least 50%, usually at least 80%, at least
90% or all of the PDZ proteins disclosed herein as being expressed in
hematopoietic cells (see Table 6).

[0231]In an embodiment the array includes at least one, preferably at
least 5 and sometimes all of the following PDZ proteins present in
lymphocytes: BAI I associated prot., Connector enhancer, DLG5 (pdlg),
DVL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot.,
KIAA147, KIAA0300, KIAA0380, KIAA0440, KIAA0545, KIAA0807, KIAA0858,
KIAA0902, novel serine protease, PDZK1, PICK8, PTN-3, RPIP8, serine
protease, 26s subunit p27, hSYNTENIN, TAX1-IP, TAX2-like protein, wwp3,
X11 prot. beta, ZO1. In this context, "array" refers to an ordered series
of immobilized polypeptides in which the identity of each polypeptide is
associated with its location. In some embodiments the plurality of
polypeptides are arrayed in a "common" area such that they can be
simultaneously exposed to a solution (e.g., containing a ligand or test
agent). For example, the plurality ofpolypeptides can be on a slide,
plate or similar surface, which may be plastic, glass, metal, silica,
beads or other surface to which proteins can be immobilized. In a
different embodiment, the different immobilized polypeptides are situated
in separate areas, such as different wells of multi-well plate (e.g., a
24-well plate, a 96-well plate, a 384 well plate, and the like). It will
be recognized that a similar advantage can be obtained by using multiple
arrays in tandem.

[0232]a) Analysis of PDZ-PL Inhibition Profile

[0233]In one aspect, the invention provides a method for determining if a
test compound inhibits any PDZ-ligand interaction in large set of
PDZ-ligand interaction (e.g., some or all of the PDZ-ligands interactions
described in Table 2; a majority of the PDZ-ligands identified in a
particular cell or tissue as described supra (e.g., lymphocytes) and the
like. In one embodiment, the PDZ domains of interest are expressed as
GST-PDZ fusion proteins and immobilized as described herein. For each PDZ
domain, a labeled ligand that binds to the domain with a known affinity
is identified as described herein.

[0234]As disclosed herein, numerous PDZ-PL interactions occur in cells of
the hematopoietic system. For any known or suspected modulator (e.g.,
inhibitor) of a PDL-PL interaction(s), it is useful to know which
interactions are inhibited (or augmented). For example, an agent that
inhibits all PDZ-PL interactions in a cell (e.g., a lymphocyte) will have
different uses than an agent that inhibits only one, or a small number,
of specific PDZ-PL interactions. The profile of PDZ interactions
inhibited by a particular agent is referred to as the "inhibition
profile" for the agent, and is described in detail below. The profile of
PDZ interactions enhanced by a particular agent is referred to as the
"enhancement profile" for the agent. It will be readily apparent to one
of skill guided by the description of the inhibition profile how to
determine the enhancement profile for an agent. The present invention
provides methods for determining the PDZ interaction
(inhibition/enhancement) profile of an agent in a single assay.

[0235]In one aspect, the invention provides a method for determining the
PDZ-PL inhibition profile of a compound by providing (i) a plurality of
different immobilized polypeptides, each of said polypeptides comprising
a PDZ domain and a non-PDZ domain and (ii) a plurality of corresponding
ligands, wherein each ligand binds at least one PDZ domain in (i), then
contacting each of said immobilized polypeptides in (i) with a
corresponding ligand in (ii) in the presence and absence of a test
compound, and determining for each polypeptide-ligand pair whether the
test compound inhibits binding between the immobilized polypeptide and
the corresponding ligand.

[0236]Typically the plurality is at least 5, and often at least 25, or at
least 40 different PDZ proteins. In a preferred embodiment, the plurality
of different ligands and the plurality of different PDZ proteins are from
the same tissue or a particular class or type of cell, e.g., a
hematopoietic cell, a lymphocyte, a neuron and the like. In a most
preferred embodiment, the plurality of different PDZs represents a
substantial fraction (e.g., at least 80%) of all of the PDZs known to be,
or suspected of being, expressed in the tissue or cell(s), e.g., all of
the PDZs known to be present in lymphocytes (for example, at least 80%,
at least 90% or all of the PDZs disclosed herein as being expressed in
hematopoietic cells).

[0237]In one embodiment, the inhibition profile is determined as follows:
A plurality (e.g., all known) PDZ domains expressed in a cell (e.g.,
lymphocytes) are expressed as GST-fusion proteins and immobilized without
altering their ligand binding properties as described supra. For each PDZ
domain, a labeled ligand that binds to this domain with a known affinity
is identified. If the set of PDZ domains expressed in lymphocytes is
denoted by {P1 . . . Pn}, any given PDZ domain Pi binds a (labeled)
ligand Li with affinity Kdi. To determine the inhibition profile for
a test agent "compound X" the "G" assay (supra) can be performed as
follows in 96-well plates with rows A-H and columns 1-12. Column 1 is
coated with P1 and washed. The corresponding ligand L1 is added to
each washed coated well of column 1 at a concentration 0.5 Kd1 with
(rows B, D, F, H) or without (rows A, C, E, F) between about 1 and about
1000 μM) of test compound X. Column 2 is coated with P2, and L2
(at a concentration 0.5 Kd2) is added with or without inhibitor X.
Additional PDZ domains and ligands are similarly tested.

[0238]Compound X is considered to inhibit the binding of Li to Pi if the
average signal in the wells of column i containing X is less than half
the signal in the equivalent wells of the column lacking X. Thus, in this
single assay one determines the full set of lymphocyte PDZs that are
inhibited by compound X.

[0239]In some embodiments, the test compound X is a mixture of compounds,
such as the product of a combinatorial chemistry synthesis as described
supra. In some embodiments, the test compound is known to have a desired
biological effect, and the assay is used to determine the mechanism of
action (i.e., if the biological effect is due to modulating a PDZ-PL
interaction).

[0240]It will be apparent that an agent that modulates only one, or a few
PDZ-PL interactions, in a panel (e.g., a panel of all known PDZs
lymphocytes, a panel of at least 10, at least 20 or at least 50 PDZ
domains) is a more specific modulator than an agent that modulate many or
most interactions. Typically, an agent that modulates less than 20% of
PDZ domains in a panel (e.g., Table 2) is deemed a "specific" inhibitor,
less than 6% a "very specific" inhibitor, and a single PDZ domain a
"maximally specific" inhibitor.

[0241]It will also be appreciated that "compound X" may be a composition
containing mixture of compounds (e.g., generated using combinatorial
chemistry methods) rather than a single compound.

[0242]Several variations of this assay are contemplated:

[0243]In some alternative embodiments, the assay above is performed using
varying concentrations of the test compound X, rather than fixed
concentration. This allows determination of the Ki of the X for each PDZ
as described above.

[0244]In an alternative embodiment, instead of pairing each PDZ Pi with a
specific labeled ligand Li, a mixture of different labeled ligands is
created that such that for every PDZ at least one of the ligands in the
mixture binds to this PDZ sufficiently to detect the binding in the "G"
assay. This mixture is then used for every PDZ domain.

[0245]In one embodiment, compound X is known to have a desired biological
effect, but the chemical mechanism by which it has that effect is
unknown. The assays of the invention can then be used to determine if
compound X has its effect by binding to a PDZ domain.

[0246]In one embodiment, PDZ-domain containing proteins are classified in
to groups based on their biological function, e.g. into those that
regulate chemotaxis versus those that regulate transcription. An optimal
inhibitor of a particular function (e.g., including but not limited to an
anti-chemotactic agent, an anti-T cell activation agent, cell-cycle
control, vesicle transport, apoptosis, etc.) will inhibit multiple
PDZ-ligand interactions involved in the function (e.g., chemotaxis,
activation) but few other interactions. Thus, the assay is used in one
embodiment in screening and design of a drug that specifically blocks a
particular function. For example, an agent designed to block chemotaxis
might be identified because, at a given concentration, the agent inhibits
2 or more PDZs involved in chemotaxis but fewer than 3 other PDZs, or
that inhibits PDZs involved in chemotaxis with a Ki>10-fold better
than for other PDZs. Thus, the invention provides a method for
identifying an agent that inhibits a first selected PDZ-PL interaction or
plurality of interactions but does not inhibit a second selected PDZ-PL
interaction or plurality of interactions. The two (or more) sets of
interactions can be selected on the basis of the known biological
function of the PDZ proteins, the tissue specificity of the PDZ proteins,
or any other criteria. Moreover, the assay can be used to determine
effective doses (i.e., drug concentrations) that result in desired
biological effects while avoiding undesirable effects.

[0247]b) Side Effects of PDZ-PL Modulator Interactions

[0248]In a related embodiment, the invention provides a method for
determining likely side effects of a therapeutic that inhibits PDZ-ligand
interactions. The method entails identifying those target tissues, organs
or cell types that express PDZ proteins and ligands that are disrupted by
a specified inhibitor. If, at a therapeutic dosage, a drug intended to
have an effect in one organ system (e.g., hematopoietic system) disrupts
PDZ-PL interactions in a different system (e.g., CNS) it can be predicted
that the drug will have effects ("side effects") on the second system. It
will be apparent that the information obtained from this assay will be
useful in the rational design and selection of drugs that do not have the
side-effect.

[0249]In one embodiment, for example, a comprehensive PDZ protein set is
obtained. A "perfectly comprehensive" PDZ protein set is defined as the
set of all PDZ proteins expressed in the subject animal (e.g., humans). A
comprehensive set may be obtained by analysis of, for example, the human
genome sequence. However, a "perfectly comprehensive" set is not required
and any reasonably large set of PDZ domain proteins (e.g., the set of all
known PDZ proteins; or the set listed in Table 6) will provide valuable
information.

[0250]In one embodiment, the method involves some of all of the following
steps:

[0251]a) For each PDZ protein, determine the tissues in which it is highly
expressed. This can be done experimentally although the information
generally will be available in the scientific literature;

[0252]b) For each PDZ protein (or as many as possible), identify the
cognate PL(s) bound by the PDZ protein;

[0253]c) Determine the Ki at which the test agent inhibits each PDZ-PL
interaction, using the methods described supra;

[0254]d) From this information it is possible to calculate the pattern of
PDZ-PL interactions disrupted at various concentrations of the test agent

By correlating the set of PDZ-PL interactions disrupted with the
expression pattern of the members of that set, it will be possible to
identify the tissues likely affected by the agent.

[0255]Additional steps can also be carried out, including determining
whether a specified tissue or cell type is exposed to an agent following
a particular route of administration. This can be determined using basis
pharmacokinetic methods and principles.

Modulation of Activities

[0256]The PDZ binding moieties and PDZ protein-PL protein binding
antagonists of the invention are used to modulate biological activities
or functions of cells (e.g., hematopoietic cells, such as T cells and B
cells and the like), endothelial cells, and other immune system cells, as
described herein, and for treatment of diseases and conditions in human
and nonhuman animals (e.g., experimental models). Exemplary biological
activities are listed supra.

[0258]The PL proteins and PDZ proteins listed in TABLE 2 are well
characterized, and one of skill, guided by this disclosure (including the
discovery of the interactions between PL proteins and PDZ proteins
described herein), will recognize many uses for modulators (e.g.,
enhancers or inhibitors) of PDZ-PL interactions such as those described
in TABLE 2. To further assist the reader, a discussion of the
characteristics of selected PL proteins (and their function) is provided
infra. It will be recognized that this discussion is not comprehensive
and is not intended to limit the invention in any way. Moreover, nothing
in this section should be construed as an intention by the inventors to
be limited to a particular mechanism of action.

A. CD6

[0259]As shown supra, CD6 binds PDZ protein 41.8. CD6 is expressed on
thymocytes, T cells, and B cell chronic lymphocytic leukemias. CD6 plays
a role in T cell co-stimulation and CD6 negative T cells are less
autoreactive than CD6 positive T cells. Inhibition of CD6 and CD6/41.8
interactions is predicted to reduce the symptoms of graft-versus-host
disease (GVHD) or psoriasis. Thus, in one embodiment of the invention,
GVHD is reduced in a patient receiving donor bone marrow cells by
pre-treating the cells with an effective amount of an antagonist. In
combination with post-transplantation immunosuppressive therapy such as
FK506, Cellcept, or cyclosporin, CD6-PDZ interaction inhibitors will
improve overall survival of transplantation patients (e.g., leukemia
patients).

B. CD49e (ALPHA-4)

[0260]As shown by the experiments reported herein, the C-terminal end of
CD49e binds to the PDZ-domain-containing protein 41.8 kD. CD49e is a 110
kD transmembrane membrane protein of the integrin alpha family (integrin
alpha 5). Paired with the integrin beta-1 subunit it forms VLA-5. VLA-5
is expressed predominantly on hematopoietic and lymphoid lineage cells
including monocytes, basophils, T cells, and activated B cells. VLA-5 is
the receptor for the ubiquitously-expressed adhesion molecule
fibronectin. Tissue injury such as myocardial infarction releases soluble
fragments of fibronectin. Binding of these soluble fragments to VLA-5
results in chemotaxis of immune cells including monocytes to the source
of fibronectin, as well as down-modulation of VLA-5 expression on these
cells. Such ligand-induced down-modulation is a common and required
feature of chemotaxic receptors. Once immune cells migrate fully to the
source of fibronectin, adhesion to the fibronectin surface is enhanced by
fibronectin-VLA-5 interaction. Without intending to be bound by a
particular mechanism, the 41.8/CD49e interaction is believed to be
necessary for proper membrane distribution of CD49e and/or recycling of
CD49e such that when it is disrupted, the migration and adherence to
fibronectin-containing surfaces is similarly disrupted, resulting in an
inability of immune system cells to effectively migrate toward a
fibronectin source and adhere to fibronectin-containing surfaces. Such
disruption would therefore result in desirable reduced inflammatory
processes, including reduced post-myocardial infarction inflammation.
Other diseases to be treated include but are not limited to joint
inflammation, psoriasis, contact allergy, Crohn's Disease, inflammatory
bowel disease, eczema, atopic dermatitis.

C. CD49F (VLA-6 α Subunit)

[0261]As shown supra, CD49F binds PDZ protein 41.8. CD49f is known as an
integrin subunit that pairs either with the β1 integrin subunit
(CD29), forming VLA-6, or with CD104 (β4 integrin subunit). The
integrin supergene family consists of a number of cell surface
αβ heterodimers important for many different physiologic
processes, including embryogenesis, thrombosis, wound healing,
tumorigenesis and immune responses. Each β chain can pair with
various α chains. Both VLA-6 and CD49f/CD 104 are widely expressed
on epithelia in non-lymphoid tissues. VLA-6 is also expressed on
platelets, monocytes, thymocytes and T lymphocytes, with an increased
expression on activated and resting memory T cells.

[0262]Inhibition of interactions between VLA-6 and 41.8 has a number of
therapeutic functions such as the prevention and treatment of metastatic
cancers, and treatment of overactive immunity. For example, VLA-6 is
associated with invasivion of prostrate carcinoma and plays a role in the
metastasis of breast cancer. Blockage of VLA-6 function combined with
conventional treatment for prostrate cancer, would be a more effective
treatment by preventing metastatic disease (see, Cress et al., 1995,
Cancer Metastasis R). Blockage of CD49f through PDZ interaction may also
treat Rh incompatibility by blunting memory response or in the treatment
of keloids.

D. CD138 (Syndecan-1)

[0263]CD138 is a transmembrane proteoglycan receptor with the
extracellular domain functioning as a ligand binding domain for various
extracellular matrix components and the intracellular portion functioning
to alter cytoskeleton and transduce intracellular signals. CD138 also
binds FGF2 and may be a co-receptor for FGF receptor (Yayon et al.,
1991).

[0264]As shown supra, CD138 interacts with 41.8 kD protein and TIAM1. The
c-terminus of CD138 has also been reported to bind the PDZ domains of
syntenin and human CASK (Cohen et al., 1998, J. Cell. Biol. 142:129-138;
Grootjans et al., 1997, PNAS 94:13683-13688; Hsueh et al., 1998, J. Cell.
Biol. 142:139-151). CD138 is expressed in pre-B cells, immature B cells,
plasma cells, neural cells, the basolateral surface of epithelial cells,
embryonic mesenchymal cells, vascular smooth muscle cells, endothelial
cells and neural cells but not mature circulating B cells. The
interaction between CD138 and the PDZ domains of the 41.8 kD protein and
TIAM1 proteins is believed to be necessary for the proper distribution of
CD138 on the cell surface. Disruption of the interaction by
administration of an effective amount of an antagonist is expected to
interfere with the migration and adherence of cells to the extracellular
matrix, resulting in reduced inflammatory and humoral immune responses.
Inhibition of CD138 may be used to treat without limitation diseases such
as post-myocardial infarction inflamatory damage, joint injury,
rheumatoid arthritis, vasculitis, drug reaction, scleraderma, SLE,
Hashimoto thyroiditis, Goodpasture's syndrome, juvenile insulin-dependent
diabetes, psoriasis.

E. CD98

[0265]As shown supra, CD98 interacts with MPP2. CD98 is expressed at high
levels on monocytes and at low levels on T cells, B cells, splenocytes,
NK cells, and granulocytes. CD98 plays roles in adhesion, fusion and is a
L-type amino acid transporter. CD98 is also involved in virus-mediated
cell fusion (e.g. paramyoviruses: parainfluenza virus type 2, Newcastle
disease virus, and rubulaviruses) and antagonism of CD98 function is
expected to treat) viral infections and limit viral spread. CD98
inhibitors can be an antiviral agent for, but not limited to
paramyovirus, parainfluenza, Newcastle disease and rubula. Other roles
include treatment for acute leukemias.

F. CLASP-1

[0266]As shown supra, CLASP-1 interacts with DLG1, PSD95, and NeDLG.
CLASP-1 is a member of a superfamily of immune-cell associated proteins
with similar motifs (see PCT/US99/22996 published as WO 00/20434).
CLASP-1 functions in the maintenance of the immune synapse. The CLASP-1
transcript is present in lymphoid organs and neural tissue, and the
protein is expressed by T and B lymphocytes and macrophages in the MOMA-1
subregion of the marginal zone of the spleen, an area known to be
important in T:B cell interaction. CLASP-1 staining of individual T and B
cells exhibits a preactivation structural polarity, being organized as a
"ball" or "cap" structure in B cells, and forming a "ring", "ball" or
"cap" structure in T cells. The placement of these structures is adjacent
to the microtubule-organizing center ("MTOC"). CLASP-1 antibody staining
indicates that CLASP-1 is at the interface of T-B cell conjugates that
are fully committed to differentiation. Antibodies to the extracellular
domain of CLASP-1 also block T-B cell conjugate formation and T cell
activation.

[0271]As shown supra, CLASP-2 interacts with PSD-95, NeDLG, and DLG1.
CLASP-2 is a member of a superfamily of immune-cell associated proteins
with similar motifs (see copending U.S. patent Ser. No. 09/547,276 filed
Apr. 11, 2000; WO 00/10158 filed Apr. 11, 2000; WO 00/10156 filed Apr.
11, 2000). The CLASP-2 transcript is present most strongly in placenta
followed by lung, kidney and heart and the protein is expressed in T and
B cells, and kidney epithelial cells.

[0272]Inhibition of the interaction of CLASP-2 and PDZ domains will
interfere with CLASP-2 function resulting in interference with T and B
cell function (e.g., T and B cell activation), signal transduction,
cell-cell interactions, and membrane organization. In addition, since
CLASP-2 is present in heart, blocking CLASP-2 function or expression can
selectively block immune responses in the heart (for example, to
selectively stop immune response in the heart compartment, e.g.,
following cardiac transplant rejection or post-MI inflammation, without
compromising immunity elsewhere). Other diseases to be treatment by
CLASP-1 agonists/antagonists include, but is not limited to, rheumaotoid
arthritis, juvenile diabetes, organ rejection, graft-versus-host disease,
scleroderma, multiple sclerosis.

J. CD95 (Apo-1/Fas)

[0273]CD95 (Fas/Apo-1) and Fas ligand (FasL) are a receptor-ligand pair
involved in lymphocyte homeostasis and peripheral tolerance. Binding of
Fas by its ligand results in apoptotic cell death, an important major
mechanism for safe clearance of unwanted cell during resolution of the
acute inflammatory response. As is shown supra, CD95 binds the PDZ
domains DLG1, PSD95, NeDLG, and 41.8. Inhibition of the interaction of
CD95 and PDZ domains.

[0276]Since DNAM-1 itself does not have a SH3 binding domain but only has
the Src phosphorylation site at Y322, an adaptor molecule must be present
to bridge DNAM-1 and Fyn. DLG1 has been described in the literature to be
present in T cells (Hanada, et al. 1997. J Biol Chem 272:26899), but does
not bind Fyn. PSD95 does not have a SH3 binding site. However, several of
the other PDZ proteins do have SH3 binding domains including but not
limited to NeDLG, RGS12, MPP2 and can fulfill this function. These
adaptor PDZ listed supra binds to DNAM-1 through its PDZ domain and
simultaneously binds to the SH3 domain of Fyn through its proline-rich
sequences just N-terminal to the PDZ domain. The Y139 is a candidate
phosphorylation site to control association of Fyn to DNAM-1 and the
adaptor PDZ.

[0277]Based on this analysis, inhibition of PDZ association with DNAM-1
using the reagents of the invention will inhibit Fyn association with
DNAM-1 and the subsequent Y322 phosphorylation and activation of
cytotoxic T cells. Diseases that can be treated include but are not
limited to Crohn's Disease, multiple sclerosis, ulcerative colitis,
inflammatory bowel disease, graft-versus-host, juvenile diabetes,
Hashimoto's disease.

M. CD83 (HB15)

[0278]CD83 is a transmembrane glycoprotein, expressed predominantly on
activated dendritic cells (DCs), Langerhans cells in the skin, with some
weak expression detected on activated peripheral lymphocytes, and
interdigitating reticulum cells within the T cell zones of lymphoid
organs (Zhou and Tedder, 1995, J. Immunol. 154:3821-3835; Zhou et al.,
1992, J. Immunol. 149:735-742). CD83 is up-regulated de novo upon
activation of an immature DCs, and is the major discriminating marker and
characteristic for activated, mature DCs (Czerniecki et al., 1997, J.
Immunol. 159:3823-3837). DCs function as antigen presenting cells (APCs).
Upregulation and expression of CD83 thus appears to be required for DCs
to mature and function as APCs.

[0279]As shown by experiments described supra, the CD83 binds to the PDZ
domains of DLG1, PSD95, and NeDLG. These interactions between CD83 and
PDZ domains, and between CD83 and DLG1, PSD95, and NeDLG are believed to
be important for proper distribution and recycling of CD83. Disruption of
CD83 and PDZ proteins with agonists and antagonist can be used to treat,
but not limited to, psoriasis, cancers, allergies, autoimmune diseases
such as multiple sclerosis, system lupus erythematosis.

[0280]CD44 is single pass transmembrane protein that has several different
isoforms due to alternative splicing. It has a broad pattern of
expression being detected on both hematopoietic and non-hematopoietic
cell types including epithelial, endothelial, mesenchymal and neuronal
cells. CD44H is a major isoform that is expressed in lymphoid, myeloid
and erythroid cells (reviewed in Barclay et al., 1997, The Leukocyte
Antigen Facts Book, 2ed, Academic Press). CD44 is a receptor for
hyaluronate (HA), which is a constituent of the extracellular matrix
(ECM). In the immune system, CD44 functions as an adhesion molecule on
the surface of leukocytes and erythrocytes that binds HA polymers in the
ECM, and it can also act as a signaling receptor when HA becomes soluble
during inflammatory reactions or tissue damage. The cytoplasmic region of
CD44 has been shown to bind or be associated with the actin cytoskeleton
through interactions with spectrin and members of the ERM (ezrin,
radixin, and meosin) family (reviewed in Lesley et al., 1993; Bajorath,
2000, Proteins 39:103-111). Additionally, CD44 is associated with the
non-receptor tyrosine kinase p56Lck (Taher et al., 1996, J. Biol. Chem.
271:2863-2867. CD44 has been shown to be a co-stimulatory molecule with
CD3/TCR engagement to activate T cells (reviewed in Aruffo, 1996, J.
Clin. Invest. 98:2191-2192).

[0281]As described supra by experiments reported herein, the C-terminus of
CD44 is a ligand for the PDZ domain contained in MPP1, prIL-16 and MINT1.
It is believed that the interactions of CD44 with PDZ domains, and
between CD44 with MPP1, prIL-16 and MINT1 function in maintenance of
leukocyte structure and in leukocyte signaling. Thus, when a CD44-PDZ
interaction is disrupted, CD44 will fail to transduce proper
intracellular signals, and maintain proper distribution of CD44 on the
surface, which will prevent adhesion of leukocytes to the endothelium
during inflammation and tissue damage. Administration of
agonists/antagonists of this interaction will thus result in, but not
limited to, reduced inflammatory responses during tissue ischemia and
cell lysis (e.g., rhabdomyosis), vascular insufficiencies (e.g.
frostbite), psoriasis, eczema, graft-versus-host disease, granuloma
annulare, scleroderma.

[0283]According to the present invention, the interaction of CD97 with
DLG1 and the 41.8 kd protein can be altered to interfere with proper
membrane distribution of CD97 and/or recycling of CD97. Such modulation
will affect CD97 dependent adherence of cells with therapeutic benefit.
Without being limited, agonists and antagonists of CD97-PDZ protein
interaction can be used to treat rheumatoid arthritis, osteoarthritis,
Crohn's Disease, Ulcerative colitis, psoriasis.

[0285]Interactions between Glycophorin C and PDZ proteins DLG, PSD95,
NeDLG, MMP2, AF6, 41.8, and MINT1 are believed necessary for maintenance
of the physical integrity of cells in which they are expressed.
Modulation of GC-PDZ interactions will alter with the function of these
and can be utilized to treat, but not limited to, polycythemia vera,
spherocytosis.

Q. CDw128A (IL8RA)

[0286]As is described supra, CDW128A binds to the PDZ domains of DLG1 and
NeDLG. There are two forms for the IL-8 receptor, IL-8RA (CDw128A) and
IL-8RB (CDw128B) both of which are members of the G protein-coupled
receptor superfamily and chemokine receptor branch of rhodopsin family.
CDw128A and CDw128B both bind IL-8 with the same affinity but only
CDw128B, binds three other IL-8-related CXC chemokines: melanoma
growth-stimulating activity (GRO/MGSA), neutrophil-activating peptide 2
(NAP-2) and ENA-78. See, e.g., Ahuja, S K. And Murphy, P M. 1996. J Biol
Chem 271:20545-50.

[0287]CDw128A is expressed on all granulocytes, a subset of T cells,
monocytes, endothelial cells, keratinocytes, erythrocytes, and melanoma
cells. IL-8 induces chemotaxis of neutrophils, basophils, and T
lymphocytes and increases neutrophil and monocyte adhesion to endothelial
cells. The binding of IL-8 to IL8RA induces a transient increase in
intracellular calcium levels, activation of phospholipase D, a
respiratory burst of neutrophils and chemotaxis. This pro-inflammatory
response is effective in normal immune responses. Inhibitors of CDw128A
are useful for treatment of psoriasis, rheumatoid arthritis,
polyarthritis, and for control of angiogenesis-dependent disorders such
as melanomas and breast cancer.

(R) CD3-eta(η)

[0288]CD3-η a splice variant of CD3 zeta and a component of the
CD3/TCR complex, which is required for antigen recognition, signal
transduction and activation of T cells (Weiss and Littman, 1994, Cell
76:263-274). See, Barclay et al., 1997, The Leucocyte antigen facts book,
2nd Ed, Academic Press. As shown by experiments reported herein, the
C-terminal region of CD3-η is a ligand for the PDZ domains of MINT1,
41.8 protein, DLG1, and PSD95. The interactions of CD3-η with PDZ
domains, and of CD3-η with MINT1, 41.8 protein, DLG1, and PSD95 are
believed to be important activation of T cells, which is required for all
cellular immune responses. Modulation of this interaction by agonists and
antogonists can be used to treat, but is not limited to, acute and
chronic allograft rejection, multiple sclerosis, graft-versus-host
disease, rheumatoid arthritis.

(S) LPAP(CD45-AP, LSM-1)

[0289]LPAP is a transmembrane protein expressed on resting and activated
T- and B-cells. LPAP has been shown to bind to CD45, a protein that is
part of the T-cell receptor complex and has been found to co-localize
with CD4, CD2 and Thy-1. LPAP has also been co-immune precipitated with
p56(lck) and ZAP-70. The actual function of LPAP is unknown, but it has
been suggested that it is an assembly molecule for the CD45 complex.

[0290]As shown supra, LPAP binds to DLG-1 and MINT-1. Notably, DLG-1 and
MINT-1 are both expressed in T-cells. It has also been shown that DLG-1
co-precipitates with p56(lck) in T-cells. The assays described herein
also demonstrated that DLG-1 binds to CD95 and KV1.3, and binding of
MINT-1 to KV1.3. All of these molecules are involved in signaling by the
TCR. LPAP is believed to function in organizing the signaling by CD45 in
T-cell activation, possibly by recruiting p56(lck) as a substrate for
CD45. Blocking the function of CD45 has been shown to severely impair the
T-cell response. Inhibiting the interaction between LPAP and PDZ proteins
is expected to alter the CD45-mediated path from the rest of the immune
response. Agonists and antagonists of PL-PDZ binding can be used to
treat, but is not limited to, rheumatoid arthritis, transplant rejection,
multiple sclerosis, scleroderma, graft-versus-host disease.

(T) CD46 (Complement Membrane Cofactor Protein (MCP))

[0291]CD46 is a membrane protein expressed on all nucleated cells, but not
on erythrocytes. CD46 is a member of the regulator of complement
activation protein family. Its primary function is the protection of
cells from complement attack by inactivating membrane deposited C3b/C4b
complement (Liszewski, et al., 1999, Adv. Immunol. 61:201-283). CD46
exists in more than 8 isoforms that are generated by differential
splicing, with molecular weights ranging from 45 to 70 kD. In addition to
the above function, CD46 also serves as the receptor for the measles
virus and for other pathogenic microorganisms (e.g. Streptococcus
pyogenes) (Manchester et al, 1994, Proc. Natl. Acad. Sci. USA 91:2161;
Okada et al., 1995, Proc. Natl Acad. Sci. USA 92:2489-2493). CD46 also
appears to be over-expressed on certain tumors (Jurianz et al., 1999, Mol
Immunol 36:929-939) thus rendering tumor cells insensitive to the action
of complement. See, Barclay et al., (1997) The Leucocyte antigen facts
book, 2ed, Academic Press.

[0292]As shown supra, CD46 binds DLG1, PSD95 and Ne-DLG. This interaction
is believed to be necessary for proper membrane distribution of CD46
and/or recycling of CD46. Alteration of the CD46-PDZ interaction can
reduce the ability of measles virus and other pathogens to enter cells,
renders CD46-expressing tumors susceptible to attack by complement. The
administration of CD46-PDZ interaction agonists and antagonists is useful
for the treatment of, but not limited to, cancers and viral infectious
diseases.

(U) CDw128B

[0293]As is described supra, CDw128B binds to the PDZ domains of DLG1,
NeDLG, PSD95, and 41.8 in the assays described supra. There are two forms
for the IL-8 receptor, IL-8RA (CDw128A) and IL-8RB (CDw128B) both of
which are members of the G protein-coupled receptor superfamily and
chemokine receptor branch of rhodopsin family. CDw128A and CDw128B both
binds IL-8 with equal affinity but only CDw128B, also binds three other
IL-8-related CXC chemokines: melanoma growth-stimulating activity
(GRO/MGSA), neutrophil-activating peptide 2 (NAP-2) and ENA-78. See,
e.g., Ahuja, S K and Murphy, P M. 1996. J Biol Chem 271:20545-50.

[0294]CDw128B is expressed on all granulocytes, a subset of T cells,
monocytes, endothelial cells, keratinocytes, erythrocytes, and melanoma
cells. IL-8 induces chemotaxis of neutrophils, basophils, and T
lymphocytes but diminished relative to IL8RA and increases neutrophil and
monocyte adhesion to endothelial cells. The binding of IL-8A to its
receptor induces a transient increase in intracellular calcium levels and
granule release but does not induce activation of phospholipase D or a
respiratory burst in neutrophils. This pro-inflammatory response is
effective in normal immune responses. Inhibitors of CDw128B are useful
for treatment of psoriasis, rheumatoid arthritis, polyarthritis, and for
control of angiogenesis-dependent disorders such as melanomas and breast
cancer.

(V) DOCK2

[0295]The DOCK family are a group of morphogenetic transmembrane proteins
that interacts with cytoskeleton to affect cell shape changes. Members of
this new family include Drosophila myoblast city (mbc), DOCK180, DOCK2,
DOCK3, CED5, KIAA0209 and CLASP. The prototypical molecule, DOCK1 or
DOCK180 is the human homolog of C. elegans gene, CED5 which is involved
in the engulfment and phagacytosis by macrophages of apoptotic cells.
DOCK2 is found in peripheral blood lymphocytes and can convert a flatten
cell into a rounded morphology upon transfection (Nagase, et. al. 1996.
DNA Res 3:321-329, Nishihara, H. 1999. Hokkaido Igaku Zasshi 74:157).

[0296]As shown supra, DOCK2 is a PL and binds to PDZ proteins. Using PDZ
proteins as an adaptor, DOCK2 complexes with A, B, C to control cell
shape in preparation for transit of lymphocytes for vascular circulation.
Modulation of DOCK2 by agonists and antagonists of its PDZ protein
interaction can be used to treat, but is not limited to, acute leukemia,
blast crisis, post-myocardial infarction inflammation, post-traumatic
inflammation.

W. CD34

[0297]As is shown supra, CD34 binds DLG1, PSD95, and NeDLG. CD34 is
expressed on a small subpopulation of bone marrow cells which includes
hematopoietic stem cells. CD34 is also present on bone marrow stomal
cells and on endothelial cells. The selectins CD62L (L-selectin) and
CD62E (E-selectin) bind CD34. CD34 mediates attachment and rolling of
leukocytes. The hematopoietic stem cell properties of CD34 include
myeloid differentiation of stem cells. Modulation of the CD34-PDZ
interaction with agonists and/or antagonists can be used to treat, but is
not limited to, myelodysplasia, leukemias, post-traumatic inflammation,
post-myocardial infarction inflammation.

X. Fc Epsilon Receptor Beta I Chain (Fc&RβI)

[0298]The high affinity receptor for human IgE, FcεRI, is composed
of an α, β, and disulfide-linked γ homodimer. The
α-chain binds the Fc portion of IgE, whereas the β-chain
serves to amplify signals that are transduced through the γ-chain
homodimer. Both αβγ2 tetramer and αγ2 trimer
complexes exist, but the β-chain amplifies the signal 5- to 7-fold,
as measured by Syk activation and calcium mobilization. Additionally, the
FcεRβI is a PDZ ligand and is a member of the
CD20/FcεRβI receptor family. As is shown supra,
FcεRβI binds MINT1.

[0299]As the high-affinity receptor for IgE, FcβRI on basophils and
mast cells plays a central role in the initiation of allergic responses.
Signaling through the FcβRI begins by crosslinking of a multivalent
allergen bound to IgE. The result is vesicular degranulation, release of
histamine, leukotrienes and pro-inflammatory cytokines (IL-6 and
TNFα), factors responsible for the symptoms of immediate
hypersensitivity. Alteration of signaling by targeting PL/PDZ interaction
with agonists and antagonists can be used to treat, but is not limited
to, asthma, atopic dermatitis, eczema, drug reaction, mastocytosis,
urticaria, eosinophilia myalgia syndrome (Turner, H., et. al., 1999,
Nature 402 SUPP:B24).

Y. FAS Ligand (FasL)

[0300]CD95 (Fas/Apo-1) and Fas ligand (FasL) are a receptor-ligand pair
critically involved in lymphocyte homeostasis and peripheral tolerance.
Binding of Fas by its ligand results in apoptotic cell death, an
important major mechanism for safe clearance of unwanted cell during
resolution of the acute inflammatory response. FasL is mainly restricted
to activated T lymphocytes and is rapidly induced. Fas ligand is
frequently up-regulated in breast cancer, as compared with normal breast
epithelial cells and benign breast disease. As is shown supra, FasL binds
the KIAA0561 PDZ domain. The PDZ-PL modifiers are useful for treatment
of, but not limited to, tumors, e.g., tumors unresponsive to conventional
chemotherapy.

Z. CDW125 (IL5R)

[0301]As is shown supra, CDW125 binds PTN-4 and RGS12. CDW125 is an IL-5
receptor expressed on eosinophils and basophils. IL5 promotes growth and
differentiation of eosinophil precursors and actives mature eosinophils
(Takatsu et al 1994, Adv. Immunol. 57:145-190). The secreted form of
CDw125 has antagonistic properties and is able to inhibit IL-5-induced
eosinophil proliferation and differentiation. Modulation of CDW125
binding to PDZ domains may be used to treat, but is not limited to,
asthma, atopic dermatitis, eczema, drug reaction, urticaria,
mastocytosis, eosinophilia.

[0303]As shown by experiments reported herein, the C-terminal end of BLR-1
binds to the PDZ domain containing protein MINT1. Without intending to be
bound by a particular mechanism, the interaction between BLR-1 and MINT1,
and BLR-1 and PDZ domains is necessary for the proper distribution and
signaling of BLR-1 on the cell surface. When this interaction is
disrupted, the chemotactic abilities of lymphoid cells expressing BLR-1
is similarly disrupted. Such a disruption results in a reduced immune
response, interference with the ability of lymphocytes to properly
circulate and develop responses to antigen. Agonists and antagonists of
this interaction can be used to treat, but is not limited to, systemic
lupus erythematosus, scleroderma and other autoimmune diseases.

BB. CD4

[0304]CD4 is a co-receptor with the T cell receptor (TCR) involved in
antigen recognition. Both CD4 and TCR belong to the immunoglobulin
supergene family. T cell activation is enhanced by increasing the avidity
of T cells for effector and target cells. The cytoplasmic domain is
involved in signal transduction and association with the tyrosine kinase
p54lck. CD4 is expressed on most thymocytes, two-thirds of
peripheral blood T lymphocytes, monocytes and macrophages.

[0305]Human immunodeficiency virus type-1 (HIV-1) infects cells by
membrane fusion mediated by its envelope glycoproteins (gp120-gp41) and
is triggered by the interaction of CD4 and a chemokine co-receptor, CCR5
or CXCR4. Modulation of CD4-PDZ inhibitors with agonists and antagonists
can be used to treat, but is not limited to, HIV infection immediately
after exposure to HIV, rheumatoid arthritis, multiple sclerosis,
scleroderma, systemic lupus erythematosis, psoriasis.

6.5 AGONISTS AND ANTAGONISTS OF PDZ-PL INTERACTIONS

[0306]As described herein, interactions between PDZ proteins and PL
proteins in cells (e.g., hematopoietic cells, e.g., T cells and B cells)
may be disrupted or inhibited by the administration of inhibitors or
antagonists. Inhibitors can be identified using screening assays
described herein. In embodiment, the motifs disclosed herein are used to
design inhibitors. In some embodiments, the antagonists of the invention
have a structure (e.g., peptide sequence) based on the C-terminal
residues of PL-domain proteins listed in TABLE 2. In some embodiments,
the antagonists of the invention have a structure (e.g., peptide
sequence) based on a PL motif disclosed herein.

[0307]The PDZ/PL antagonists and antagonists of the invention may be any
of a large variety of compounds, both naturally occurring and synthetic,
organic and inorganic, and including polymers (e.g., oligopeptides,
polypeptides, oligonucleotides, and polynucleotides), small molecules,
antibodies, sugars, fatty acids, nucleotides and nucleotide analogs,
analogs of naturally occurring structures (e.g., peptide mimetics,
nucleic acid analogs, and the like), and numerous other compounds.
Although, for convenience, the present discussion primarily refers
antagonists of PDZ-PL interactions, it will be recognized that PDZ-PL
interaction agonists can also be use in the methods disclosed herein.

[0308]In one aspect, the peptides and peptide mimetics or analogues of the
invention contain an amino acid sequence that binds a PDZ domain in
hematopoietic cells such as T cells and B cells, or otherwise inhibits
the association of PL proteins and PDZ proteins. In one embodiment, the
antagonists comprise a peptide that has a sequence corresponding to the
carboxy-terminal sequence of a PL protein listed in TABLE 2, e.g., a
peptide listed TABLE 4. Typically, the peptide comprises at least the
C-terminal four (4) residues of the PL protein, and often the inhibitory
peptide comprises more than four residues (e.g., at least five, six,
seven, eight, nine, ten, twelve or fifteen residues) from the PL protein
C-terminus. See, e.g. Section 6.5.1, infra. Moreover, the C-terminal
domains of specific surface receptors expressed by hematopoietic system
and endothelial cells may themselves be used as inhibitors, and may be
used as the basis for rational design of non-peptide inhibitors. See
Section 6.6, infra.

[0311]In some embodiments, the antagonist is a peptide mimetic of a PL
C-terminal sequence. See, e.g. Section 6.5.4, infra.

[0312]In some embodiments, the inhibitor is a small molecule (i.e., having
a molecular weight less than 1 kD). See, e.g. Section 6.5.5, infra.

[0313]6.5.1 Peptide Antagonists

[0314]In one embodiment, the antagonists comprise a peptide that has a
sequence of a PL protein carboxy-terminus listed in TABLE 2. The peptide
comprises at least the C-terminal two (2) residues of the PL protein, and
typically, the inhibitory peptide comprises more than two residues (e.g,
at least three, four, five, six, seven, eight, nine, ten, twelve or
fifteen residues) from the PL protein C-terminus. Most often, the
residues shared by the inhibitory peptide with the PL protein are found
at the C-terminus of the peptide. However, in some embodiments, the
sequence is internal. Similarly, in some cases, the inhibitory peptide
comprises residues from a PL sequence that is near, but not at the
c-terminus of a PL protein (see, Gee et al., 1998, J Biological Chem.
273:21980-87).

[0315]For example, the "core PDZ motif sequence" of a hematopoietic cell
surface receptor at its C-terminus contains the last four amino acids,
this sequence may be used to target PDZ domains in hematopoietic cells.
The four amino acid core of a PDZ motif sequence may contain additional
amino acids at its amino terminus to further increase its binding
affinity and/or stability. In one embodiment, the PDZ motif sequence
peptide can be from four amino acids up to 15 amino acids. It is
preferred that the length of the sequence to be 6-10 amino acids. More
preferably, the PDZ motif sequence contains 8 amino acids. Additional
amino acids at the amino terminal end of the core sequence may be derived
from the natural sequence in each hematopoietic cell surface receptor or
a synthetic linker. The additional amino acids may also be conservatively
substituted. When the third residue from the C-terminus is S, T or Y,
this residue may be phosphorylated prior to the use of the peptide.

[0316]In some embodiments, the peptide and nonpeptide inhibitors of the
are small, e.g., fewer than ten amino acid residues in length if a
peptide. Further, it is reported that a limited number of ligand amino
acids directly contact the PDZ domain (generally less than eight) (Kozlov
et al., 2000, Biochemistry 39, 2572; Doyle et al., 1996, Cell 85, 1067)
and that peptides as short as the C-terminal three amino acids often
retain similar binding properties to longer (>15) amino acids peptides
(Yanagisawa et al., 1997, J. Biol. Chem. 272, 8539).

[0317]FIGS. 3A-H show the use of peptides to inhibit PL-PDZ interactions
using the G assay described supra. In FIGS. 3A and B, the inhibition
assays were carried out using GST fusion proteins containing PDZ domains
from DLG1 or PSD95 (see supra and TABLE 3). Binding of biotinylated PL
peptides for CLASP-2, CD46, Fas, or KV1.3 (as listed in TABLE 4) was
determined in the presence of various competitor peptides (at a
concentration of 100 μM) or in the absence of a competitor (equalized
as 100% binding). The competitor peptides were 8-mers peptides having the
sequence of C-terminus of CLASP-2 (MTSSSSVV (SEQ ID NO:227)), CD46
(REVKFTSL (SEQ ID NO:113)), or Fas (RNEIQSLV (SEQ ID NO:83)), a unlabeled
19-mer having the sequence of c-terminus of KV1.3 (i.e., non-biotinylated
AA33L as listed in TABLE 4), or a peptide having the sequence of residues
64-76 of hemoglobin (Vidal et al., 1999, J. Immunol. 163, 4811), i.e., an
unrelated competitor. The binding of biotinylated peptide (10 μM for
Fas and KV1.3, 20 uM for CLASP-2 and CD46) to GST alone was subtracted
from the binding to the fusion proteins to obtain the net signal for each
experimental condition. This net signal was then normalized by dividing
by the signal in the absence of competitor peptide and the data were
plotted. Error bars indicated the standard deviation of duplicate
measurements. Specific inhibition of CLASP-2 PL-DLG PDZ binding was
observed with the CLASP-2 8-mer, the CD46 8-mer, the Fas 8-mer, and the
KV1.3 peptide, but not in the absence of peptide or using an unrelated
peptide.

[0318]FIGS. 3C-F show similar assays using shorter peptides to inhibit
(e.g., a 3-mer and a 5-mer). FIGS. 3C-E show binding of biotinylated PL
peptides for CLASP-2, CD46, Fas, or KV1.3, at the indicated concentration
(as listed in TABLE 3) to GST fusion proteins containing PDZ domains from
NeDLG, DLG1, or PSD95 in the absence or presence of 1 mM 3-mer peptide
having the sequence of the C-terminus of CLASP-2 (SVV) (Table 3). FIG. 3F
shows the effect on binding of a 5-mer CD49E peptide (ATSDA (SEQ ID
NO:25)) to GST fusion proteins containing a PDZ domain from 41.8 Kd

[0319]6.5.2 Peptide Variants

[0320]Having identified PDZ binding peptides and PDZ-PL interaction
inhibitory sequences, variations of these sequences can be made and the
resulting peptide variants can be tested for PDZ domain binding or PDZ-PL
inhibitory activity. In embodiments, the variants have the same or a
different ability to bind a PDZ domain as the parent peptide. Typically,
such amino acid substitutions are conservative, i.e., the amino acid
residues are replaced with other amino acid residues having physical
and/or chemical properties similar to the residues they are replacing.
Preferably, conservative amino acid substitutions are those wherein an
amino acid is replaced with another amino acid encompassed within the
same designated class.

[0321]6.5.3 Peptide Mimetics

[0322]Having identified PDZ binding peptides and PDZ-PL interaction
inhibitory sequences, peptide mimetics can be prepared using routine
methods, and the inhibitory activity of the mimetics can be confirmed
using the assays of the invention. Thus, in some embodiments, the
antagonist is a peptide mimetic of a PL C-terminal sequence. The skilled
artisan will recognize that individual synthetic residues and
polypeptides incorporating mimetics can be synthesized using a variety of
procedures and methodologies, which are well described in the scientific
and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman
et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptides incorporating
mimetics can also be made using solid phase synthetic procedures, as
described, e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426. Mimetics
of the invention can also be synthesized using combinatorial
methodologies. Various techniques for generation of peptide and
peptidomimetic libraries are well known, and include, e.g., multipin, tea
bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol.
Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119;
Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol.
267:220-234.

[0323]6.5.4 Small Molecules

[0324]In some embodiments, the inhibitor is a small molecule (i.e., having
a molecular weight less than 1 kD). Methods for screening small molecules
are well known in the art and include those described supra at Section
6.4.

6.6 CELL SURFACE RECEPTORS AND PDZ-DOMAIN BINDING SEQUENCES

[0325]The following sections describe specific surface receptors expressed
by different cell types in the hematopoietic and immune response system.
The C-termini of these receptors are used as inhibitors, or serve as the
basis for designing PDZ motif sequence peptides, variants, fusion
proteins, peptidomimetics, and small molecules for use in inhibiting
PDZ-PL interactions. In a preferred embodiment, the peptides are tested
in an assay of the invention for inhibitory or modulatory activity (also
see, TABLE 4, and discussion supra).

[0358]A number of surface receptors expressed by NK cells contain a PDZ
domain motif sequence. These molecules include, but are not limited to
CD38, CD56, CD98 and DNAM-1. The specific motif sequences of CD38, CD98
and DNAM-1 have been described in the preceding paragraphs.

[0371]A number of surface receptors expressed by granulocytes contain a
PDZ domain motif sequence. These molecules include, but are not limited
to CD53, CD95, CD97, CD98, CD148, CDw125, CDw128, FcεRIβ and
G-CSFR. The specific motif sequences of most of these molecules have been
described in the preceding paragraphs.

[0374]While endothelial cells are not hematopoietic cells, they closely
interact with the hematopoietic system as they form the lining of blood
vessels. As such, endothelial cells come in contact with the cells of the
hematopoietic system. Thus, the ability to regulate endothelial cell
function provides for indirect regulation of hematopoietic cells. A
number of surface receptors expressed by endothelial cells contain a PDZ
domain motif sequence. These molecules include, but are not limited to
CD34, CD46, CD66b, CD66c, CD105, CD106, CD62e (E-selectin) and VCAM1.

[0382]FcεRIβ, CDw125, CDw128 and IL-8RB are transmembrane
receptors expressed by mast cells, basophils and eosinophils. These
receptors play a role in the activation of these cells to result in
degranulation and histamine release in allergic reactions. The C-terminal
core sequence of FcεRIβ is PIDL (SEQ ID NO:129). When
naturally-occurring residues are added to the core sequence, PPIDL (SEQ
ID NO:130), SPPIDL (SEQ ID NO:131), MSPPIDL (SEQ ID NO:132) and EMSPPIDL
(SEQ ID NO:133) may also be used to target a PDZ domain-containing
protein in mast cells. In addition, the residue E may be substituted with
G to increase its binding affinity.

[0392]The peptides of the invention or analogues thereof, may be prepared
using virtually any art-known technique for the preparation of peptides
and peptide analogues. For example, the peptides may be prepared in
linear form using conventional solution or solid phase peptide syntheses
and cleaved from the resin followed by purification procedures
(Creighton, 1983, Protein Structures And Molecular Principles, W.H.
Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides
described herein are well known in the art. The composition of the
synthetic peptides may be confirmed by amino acid analysis or sequencing
(e.g., the Edman degradation procedure and mass spectroscopy).

[0393]In addition, analogues and derivatives of the peptides can be
chemically synthesized. The linkage between each amino acid of the
peptides of the invention may be an amide, a substituted amide or an
isostere of amide. Nonclassical amino acids or chemical amino acid
analogues can be introduced as a substitution or addition into the
sequence. Non-classical amino acids include, but are not limited to, the
D-isomers of the common amino acids, α-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,
ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, α-alanine, fluoro-amino acids,
designer amino acids such as β-methyl amino acids, Cα-methyl
amino acids, Nα-methyl amino acids, and amino acid analogues in
general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).

[0394]6.7.2. Recombinant Synthesis

[0395]If the peptide is composed entirely of gene-encoded amino acids, or
a portion of it is so composed, the peptide or the relevant portion may
also be synthesized using conventional recombinant genetic engineering
techniques. For recombinant production, a polynucleotide sequence
encoding a linear form of the peptide is inserted into an appropriate
expression vehicle, i.e., a vector which contains the necessary elements
for the transcription and translation of the inserted coding sequence, or
in the case of an RNA viral vector, the necessary elements for
replication and translation. The expression vehicle is then transfected
into a suitable target cell which will express the peptide. Depending on
the expression system used, the expressed peptide is then isolated by
procedures well-established in the art. Methods for recombinant protein
and peptide production are well known in the art (see, e.g., Maniatis et
al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y.; and Ausubel et al, 1989, Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley Interscience, N.Y.).

[0397]The expression elements of the expression systems vary in their
strength and specificities. Depending on the host/vector system utilized,
any of a number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used in the
expression vector. For example, when cloning in bacterial systems,
inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac
(ptrp-lac hybrid promoter) and the like may be used; when cloning in
insect cell systems, promoters such as the baculovirus polyhedron
promoter may be used; when cloning in plant cell systems, promoters
derived from the genome of plant cells (e.g., heat shock promoters; the
promoter for the small subunit of RUBISCO; the promoter for the
chlorophyll a/b binding protein) or from plant viruses (e.g., the
35S RNA promoter of CaMV; the coat protein promoter of TMV) may be
used; when cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5 K promoter) may be used; when generating cell lines that contain
multiple copies of expression product, SV40-, BPV- and EBV-based vectors
may be used with an appropriate selectable marker.

[0399]In one insect expression system that may be used to produce the
peptides of the invention, Autographa californica nuclear polyhidrosis
virus (AcNPV) is used as a vector to express the foreign genes. The virus
grows in Spodoptera frugiperda cells. A coding sequence may be cloned
into non-essential regions (for example the polyhedron gene) of the virus
and placed under control of an AcNPV promoter (for example, the
polyhedron promoter). Successful insertion of a coding sequence will
result in inactivation of the polyhedron gene and production of
non-occluded recombinant virus (i.e., virus lacking the proteinaceous
coat coded for by the polyhedron gene). These recombinant viruses are
then used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;
Smith, U.S. Pat. No. 4,215,051). Further examples of this expression
system may be found in Current Protocols in Molecular Biology, Vol. 2,
Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

[0400]In mammalian host cells, a number of viral based expression systems
may be utilized. In cases where an adenovirus is used as an expression
vector, a coding sequence may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite leader sequence. This chimeric gene may then be inserted in
the adenovirus genome by in vitro or in vivo recombination. Insertion in
a non-essential region of the viral genome (e.g., region E1 or E3) will
result in a recombinant virus that is viable and capable of expressing
peptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.
Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter
may be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA
79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et
al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

[0401]Other expression systems for producing linear peptides of the
invention will be apparent to those having skill in the art.

[0402]6.7.3. Purification of the Peptides and Peptide Analogues

[0403]The peptides and peptide analogues of the invention can be purified
by art-known techniques such as high performance liquid chromatography,
ion exchange chromatography, gel electrophoresis, affinity chromatography
and the like. The actual conditions used to purify a particular peptide
or analogue will depend, in part, on factors such as net charge,
hydrophobicity, hydrophilicity, etc., and will be apparent to those
having skill in the art. The purified peptides can be identified by
assays based on their physical or functional properties, including
radioactive labeling followed by gel electrophoresis, radioimmuno-assays,
ELISA, bioassays, and the like.

[0404]For affinity chromatography purification, any antibody which
specifically binds the peptides or peptide analogues may be used. For the
production of antibodies, various host animals, including but not limited
to rabbits, mice, rats, etc., may be immunized by injection with a
peptide. The peptide may be attached to a suitable carrier, such as BSA
or KLH, by means of a side chain functional group or linkers attached to
a side chain functional group. Various adjuvants may be used to increase
the immunological response, depending on the host species, including but
not limited to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants such as
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0405]Monoclonal antibodies to a peptide may be prepared using any
technique which provides for the production of antibody molecules by
continuous cell lines in culture. These include but are not limited to
the hybridoma technique originally described by Koehler and Milstein,
1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et
al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad.
Sci. U.S.A. 80:2026-2030 and the EBV-hybridoma technique (Cole et al.,
1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96 (1985)). In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al, 1984, Proc. Natl. Acad. Sci.
U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological activity can be
used. Alternatively, techniques described for the production of single
chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
peptide-specific single chain antibodies.

[0406]Antibody fragments which contain deletions of specific binding sites
may be generated by known techniques. For example, such fragments include
but are not limited to F(ab')2 fragments, which can be produced by
pepsin digestion of the antibody molecule and Fab fragments, which can be
generated by reducing the disulfide bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed
(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired specificity
for the peptide of interest.

[0407]The antibody or antibody fragment specific for the desired peptide
can be attached, for example, to agarose, and the antibody-agarose
complex is used in immunochromatography to purify peptides of the
invention. See, Scopes, 1984, Protein Purification: Principles and
Practice, Springer-Verlag New York, Inc., NY, Livingstone, 1974, Methods
Enzymology Immunoaffinity Chromatography of Proteins 34:723-731.

6.8. USES OF PDZ DOMAIN BINDING AND ANTAGONIST COMPOUNDS

[0408]In one aspect of the invention, the PDZ domain binding and PDZ-PL
inhibitory compounds of the present invention are useful in regulating
diverse activities of hematopoietic cells (e.g., T cells and B cells) and
other cells involved in the immune response.

[0409]In one embodiment of the invention, the compounds of the invention
are used to inhibit leukocyte activation, which is manifested in
measurable events including but not limited to, cytokine production, cell
adhesion, expansion of cell numbers, apoptosis and cytotoxicity. As a
corollary, the compounds of the invention may be used to treat diverse
conditions associated with undesirable leukocyte activation, including
but not limited to, acute and chronic inflammation, graft-versus-host
disease, transplantation rejection, hypersensitivities and autoimmunity
such as multiple sclerosis, rheumatoid arthritis, peridontal disease,
systemic lupus erythematosis, juvenile diabetes mellitis,
non-insulin-dependent diabetes, and allergies, and other conditions
listed herein (see, e.g., Section 6.4, supra).

[0410]Thus, the invention also relates to methods of using such
compositions in modulating leukocyte activation as measured by, for
example, cytotoxicity, cytokine production, cell proliferation, and
apoptosis. Assays for activation are well known. For example, PDZ/PL
interaction antagonists can be evaluated in the following: (1) cytotoxic
T lymphocytes can be incubated with radioactively labeled target cells
and the antigen-specific lysis of these target cells detected by the
release of radioactivity, (2) helper T lymphocytes can be incubated with
antigens and antigen presenting cells and the synthesis and secretion of
cytokines measured by standard methods (Windhagen A; et al., 1995,
Immunity 2(4): 373-80), (3) antigen presenting cells can be incubated
with whole protein antigen and the presentation of that antigen on MHC
detected by either T lymphocyte activation assays or biophysical methods
(Harding et al., 1989, Proc. Natl. Acad. Sci., 86: 4230-4), (4) mast
cells can be incubated with reagents that cross-link their Fc-epsilon
receptors and histamine release measured by enzyme immunoassay
(Siraganian, et al., 1983, TIPS 4: 432-437).

[0411]Similarly, the effect of PDZ/PL interaction antagonists on products
of leukocyte activation in either a model organism (e.g., mouse) or a
human patient can also be evaluated by various methods that are well
known. For example, (1) the production of antibodies in response to
vaccination can be readily detected by standard methods currently used in
clinical laboratories, e.g., an ELISA; (2) the migration of immune cells
to sites of inflammation can be detected by scratching the surface of
skin and placing a sterile container to capture the migrating cells over
scratch site (Peters et al., 1988, Blood 72: 1310-5); (3) the
proliferation of peripheral blood mononuclear cells in response to
mitogens or mixed lymphocyte reaction can be measured using
3H-thymidine; (4) the phagocytic capacity of granulocytes,
macrophages, and other phagocytes in PBMCs can be measured by placing
PMBCs in wells together with labeled particles (Peters et al., 1988); and
(5) the differentiation of immune system cells can be measured by
labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and
measuring the fraction of the PBMCs expressing these markers.

[0413]Standard methods by which hematopoietic cells are stimulated to
undergo activation characteristic of an immune response are, for example:

[0414]A) Antigen specific stimulation of immune responses. Either
pre-immunized or naive mouse splenocytes can be generated by standard
procedures. In addition, antigen-specific T cell clones and hybridomas
(e.g., MBP-specific), and numerous B cell lymphoma cell lines (e.g.,
CH27), have been previously characterized and are available for the
assays discussed below. Antigen specific splenocytes or B-cells can be
mixed with antigen specific T-cells in the presence of antigen to
generate an immune response. This can be performed in the presence or
absence of PDZ/PL interaction antagonists to assay whether PDZ/PL
interaction antagonists modulate the immune response infra.

[0418]E) Generation of a specific T cell clone or line that recognizes a
particular antigen. A standard approach is to generate tetanus
toxin-specific T cells from a donor that has recently been boosted with
tetanus toxin. Major histocompatability complex-(MHC--) matched antigen
presenting cells and a source of tetanus toxin are used to maintain
antigen specificity of the cell line or T cell clone (Lanzavecchia, A.,
et al., 1983, Eur. J. Immun. 13: 733-738).

[0419]Assay Quantitation

[0420]The assays described above can be quantitated by a variety of well
known quantitation methods. For example:

[0421](A) Tyrosine Phosphorylation

[0422]Tyrosine phosphorylation of early response proteins such as HS1,
PLC-r, ZAP-76, and Vav is an early biochemical event following leukocyte
activation. The tyrosine phosphorylated proteins can be detected by
Western blot using antibodies against phosphorylated tyrosine residues.
Tyrosine phosphorylation of these early response proteins can be used as
a standard assay for leukocyte activation (J. Biol. Chem., 1997, 272(23):
14562-14570). Any change in the phosphorylation pattern of these or
related proteins when immune responses are generated in the presence of
potential PDZ/PL interaction antagonists is indicative of a potential
PDZ/PL interaction antagonists.

[0423](B) Intracellular Calcium Flux

[0424]The kinetics of intracellular Ca2+ concentrations are measured
over time after stimulation of cells preloaded with a calcium sensitive
dye. Upon binding Ca2+ the indicator dye (e.g., Fluor-4 (Molecular
Probes)), exhibits an increase in fluorescence level using flow
cytometry, solution fluorometry, and confocal microscopy. Any change in
the level or timing of calcium flux when immune responses are generated
in the presence of PDZ/PL interaction antagonists is indicative of an
inhibition of this response.

[0425](C) Regulation of Early Activation Markers

[0426]Increased and diminished expression/regulation of early lymphocyte
activation marker levels such as CD69, IL-2R, MHC class II, B7, and TCR
are commonly measured with fluorescently labeled antibodies using flow
cytometry. All antibodies are commercially available. Any change in the
expression levels of lymphocyte activation markers when immune responses
are generated in the presence of the PDZ/PL interaction antagonists is
indicative of an inhibition of this response.

[0427](D) Increased metabolic activity/acid release

[0428]Activation of most known signal transduction pathways trigger
increases in acidic metabolites. This reproducible biological event is
measured as the rate of acid release using a microphysiometer (Molecular
Devices), and is used as an early activation marker when comparing the
treatment of cells with potential biological therapeutics (McConnell, H.
M. et al., 1992, Science 257: 1906-1912 and McConnell, H. M., 1995, Proc.
Natl. Acad. Sci. 92: 2750-2754). Any statistically significant increase
or decrease in acid release of the PDZ/PL interaction antagonist-treated
sample, as compared to control sample (no treatment), suggest an effect
of the PDZ/PL interaction antagonist on biological function.

[0429](E) Cell Proliferation/Cell Viability Assays

[0430](1) 3H-Thimidine Incorporation

[0431]Exposure of lymphocytes to antigen or mitogen in vitro induces DNA
synthesis and cellular proliferation. The measurement of mitotic activity
by 3H-thimidine incorporation into newly synthesized DNA is one of
the most frequently used assays to quantitative T cell activation.
Depending on the cell population and form of stimulation used to activate
the T cells, mitotic activity can be measured within 24-72 hrs. in vitro,
post 3H-thimidine pulse (Mishell, B. B. and S. M. Shiigi, 1980, Selected
Methods in Cellular Immunology, W. H. Freeman and Company and Dutton, R.
W. and Pearce, J. D., 1962, Nature 194: 93). Any statistically
significant increase or decrease in CPM of the PDZ/PL interaction
antagonist-treated sample, as compared to control sample (no treatment),
suggest and effect of the PDZ/PL interaction antagonist on biological
function.

[0433](3) Bromodeoxyuridine (BrdU), athymidine analogue,
readilyincorporates into cells undergoing DNA synthesis. BrdU-pulsed
cells are labeled with an enzyme-conjugated anti-BrdU antibody (Gratzner,
H. G., 1982, Science 218:474-475). A colorimetric, soluble substrate is
used to visualize proliferating cells that have incorporated BrdU.
Reaction is stopped with sulfuric acid and plate is read at 450 nm using
a microplate reader. Any statistically significant increase or decrease
in color intensity of the PDZ/PL interaction antagonist-treated sample,
as compared to control sample (no treatment), suggest an effect of the
PDZ/PL interaction antagonist on biological function.

[0434](F) Apoptosis by Annexin V

[0435]Programmed cell death or apoptosis is an early event in a cascade of
catabolic reactions leading to cell death. A lose in the integrity of the
cell membrane allows for the binding of fluorescently conjugated
phosphatidylserine. Stained cells can be measured by fluorescence
microscopy and flow cytometry (Vermes, I., 1995, J. Immunol. Methods.
180: 39-52). In one embodiment, any statistically significant increase or
decrease in apoptotic cell number of the PDZ/PL interaction
antagonist-treated sample, as compared to control sample (no treatment),
suggest an effect of the PDZ/PL interaction antagonist on biological
function. For evaluating apoptosis in situ, assays for evaluating cell
death in tissue samples can also be used in vivo studies.

[0436](G) Quantitation of Cytokine Production

[0437]Cell supernatants harvested after cell stimulation for 16-48 hrs are
stored at -80° C. until assayed or directly tested for cytokine
production. Multiple cytokine assays can be performed on each sample.
IL-2, IL-3, IFN-γ and other cytokine ELISA Assays are available for
mouse, rat, and human (Endogen, Inc. and BioSource). Cytokine production
is measured using a standard two-antibody sandwich ELISA protocol as
described by the manufacturer. The presence of horseradish peroxidase is
detected with 3,3'5,5' tertamethyl benziidine (TMB) substrate and the
reaction is stopped with sulfuric acid. The absorbency at 450 nm is
measured using a microplate reader. Any statistically significant
increase or decrease in color intensity of the PDZ/PL interaction
antagonist-treated sample, as compared to control sample (no treatment),
suggest an effect of the PDZ/PL interaction antagonist on biological
function. See also Example 1, infra. Detection of intracellular cytokines
using anti-cytokine antibodies provides the additional advantage of
measuring cytokines fore mixed cell populations. This allows for
phenotyping measuring frequency of cytokine producing cell types in
suspension or in tissues.

[0438](H) NF-AT can be visualized by Immunostaining

[0439]T cell activation requires the import of nuclear factor of activated
T cells (NF-AT) to the nucleus. This translocation of NF-AT can be
visualized by immunostaining with anti-NF-AT antibody (Cell 1998, 93:
851-861). Therefore, NF-AT nuclear translocation has been used to assay T
cell activation. Similarly, NF-AT/luciferase reporter assays have been
used as a standard measurement of T cell activation (MCB 1996, 12:
7151-7160). Any statistically significant increase or decrease in the
nuclear translocation of NF-AT brought about by the PDZ/PL interaction
antagonist-treated sample, as compared to control sample (no treatment),
suggest an effect of the PDZ/PL interaction antagonist on biological
function. In order to optimize the use of the peptides and peptide
analogues disclosed herein in a human subject, various animal models may
be used to define certain clinical parameters. For example, the compounds
of the invention may be tested in different dosages, formulations and
route of administration in a cardiac transplant mouse model to optimize
their ability to inhibit rejection responses to solid organ transplants
(Fulmer et al., 1963, Am. J. Anat. 113:273; Jockusch et al., 1983, Exp.
Neurol. 81:749).

[0440]In situations where inhibition of a T cell response is desired, the
compounds of the inventions may be used to inhibit PDZ domain
interactions with CD3, CD4, CD6 and CDw137. In addition, the compounds of
the invention may be used to inhibit PDZ domain interactions with CD53
and CD138 in B cells. In order to inhibit IgE-mediated allergic
reactions, the compounds of the invention may be used to inhibit PDZ
domain interactions with FcεRIβ, CDw125 and CDw128.
Furthermore, a PDZ motif sequence (PL sequence) of CD95 may be used to
induce apoptosis of lymphomas.

[0443]6.9.1 Introduction of Agonists or Antagonists (e.g., Peptides and
Fusion Proteins) into Cells

[0444]In one aspect, the PDZ-PL antagonists of the invention are
introduced into a cell to modulate (i.e., increase or decrease) a
biological function or activity of the cell. Many small organic molecules
readily cross the cell membranes (or can be modified by one of skill
using routine methods to increase the ability of compounds to enter
cells, e.g., by reducing or eliminating charge, increasing lipophilicity,
conjugating the molecule to a moiety targeting a cell surface receptor
such that after interacting with the receptor). Methods for introducing
larger molecules, e.g., peptides and fusion proteins are also well known,
including, e.g., injection, liposome-mediated fusion, application of a
hydrogel, conjugation to a targeting moiety conjugate endocytozed by the
cell, electroporation, and the like).

[0445]In one embodiment, the antagonist or agent is a fusion polypeptide
or derivatized polypeptide. A fusion or derivatized protein may include a
targeting moiety that increases the ability of the polypeptide to
traverse a cell membrane or causes the polypeptide to be delivered to a
specified cell type (e.g., liver cells or tumor cells) preferentially or
cell compartment (e.g., nuclear compartment) preferentially. Examples of
targeting moieties include lipid tails, amino acid sequences such as
antennapoedia peptide or a nuclear localization signal (NLS; e.g.,
Xenopus nucleoplasmin Robbins et al., 1991, Cell 64:615).

[0446]In one embodiment of the invention, a peptide sequence or peptide
analog determined to inhibit a PDZ domain-PL protein binding, in an assay
of the invention is introduced into a cell by linking the sequence to an
amino acid sequence that facilitates its transport through the plasma
membrane (a "transmembrane transporter sequence"). The peptides of the
invention may be used directly or fused to a transmembrane transporter
sequence to facilitate their entry into cells. In the case of such a
fusion peptide, each peptide may be fused with a heterologous peptide at
its amino terminus directly or by using a flexible polylinker such as the
pentamer G-G-G-G-S (SEQ ID NO:1) repeated 1 to 3 times. Such linker has
been used in constructing single chain antibodies (scFv) by being
inserted between VH and VL (Bird et al., 1988, Science
242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:5979-5883). The linker is designed to enable the correct interaction
between two beta-sheets forming the variable region of the single chain
antibody. Other linkers which may be used include
Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:2)
(Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) and
Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp
(SEQ ID NO:3) (Bird et al., 1988, Science 242:423-426).

[0448]It is preferred that a transmembrane transporter sequence is fused
to a hematopoietic cell surface receptor carboxyl terminal sequence at
its amino-terminus with or without a linker. Generally, the C-terminus of
a PDZ motif sequence (PL sequence) must be free in order to interact with
a PDZ domain. The transmembrane transporter sequence may be used in whole
or in part as long as it is capable of facilitating entry of the peptide
into a cell.

[0449]In an alternate embodiment of the invention, a hematopoietic cell
surface receptor C-terminal sequence may be used alone when it is
delivered in a manner that allows its entry into cells in the absence of
a transmembrane transporter sequence. For example, the peptide may be
delivered in a liposome formulation or using a gene therapy approach by
delivering a coding sequence for the PDZ motif alone or as a fusion
molecule into a target cell.

[0450]The compounds of the of the invention may also be administered via
liposomes, which serve to target the conjugates to a particular tissue,
such as lymphoid tissue, or targeted selectively to infected cells, as
well as increase the half-life of the peptide composition. Liposomes
include emulsions, foams, micelles, insoluble monolayers, liquid
crystals, phospholipid dispersions, lamellar layers and the like. In
these preparations the peptide to be delivered is incorporated as part of
a liposome, alone or in conjunction with a molecule which binds to, e.g.,
a receptor prevalent among lymphoid cells, such as monoclonal antibodies
which bind to the CD45 antigen, or with other therapeutic or immunogenic
compositions. Thus, liposomes filled with a desired peptide or conjugate
of the invention can be directed to the site of lymphoid cells, where the
liposomes then deliver the selected inhibitor compositions. Liposomes for
use in the invention are formed from standard vesicle-forming lipids,
which generally include neutral and negatively charged phospholipids and
a sterol, such as cholesterol. The selection of lipids is generally
guided by consideration of, e.g., liposome size, acid lability and
stability of the liposomes in the blood stream. A variety of methods are
available for preparing liposomes, as described in, e.g., Szoka et al.,
Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,
4,501,728 and 4,837,028.

[0451]The targeting of liposomes using a variety of targeting agents is
well known in the art (see, e.g., U.S. Pat. Nos. 4,957,773 and
4,603,044). For targeting to the immune cells, a ligand to be
incorporated into the liposome can include, e.g., antibodies or fragments
thereof specific for cell surface determinants of the desired immune
system cells. A liposome suspension containing a peptide or conjugate may
be administered intravenously, locally, topically, etc. in a dose which
varies according to, inter alia, the manner of administration, the
conjugate being delivered, and the stage of the disease being treated.

[0452]In order to specifically deliver a PDZ motif sequence (PL sequence)
peptide into a specific cell type, the peptide may be linked to a
cell-specific targeting moiety, which include but are not limited to,
ligands for diverse leukocyte surface molecules such as growth factors,
hormones and cytokines, as well as antibodies or antigen-binding
fragments thereof. Since a large number of cell surface receptors have
been identified in leukocytes, ligands or antibodies specific for these
receptors may be used as cell-specific targeting moieties. For example,
interleukin-2, B7-1 (CD80), B7-2 (CD86) and CD40 or peptide fragments
there of may be used to specifically target activated T cells (The
Leucocyte Antigen Facts Book, 1997, Barclay et al. (eds.), Academic
Press). CD28, CTLA-4 and CD40L or peptide fragments there of may be used
to specifically target B cells. Furthermore, Fc domains may be used to
target certain Fc receptor-expressing cells such as monocytes.

[0453]Antibodies are the most versatile cell-specific targeting moieties
because they can be generated against any cell surface antigen.
Monoclonal antibodies have been generated against leukocyte
lineage-specific markers such as certain CD antigens. Antibody variable
region genes can be readily isolated from hybridoma cells by methods well
known in the art. However, since antibodies are assembled between two
heavy chains and two light chains, it is preferred that a scFv be used as
a cell-specific targeting moiety in the present invention. Such scFv are
comprised of VH and VL domains linked into a single polypeptide
chain by a flexible linker peptide.

[0454]The PDZ motif sequence (PL sequence) may be linked to a
transmembrane transporter sequence and a cell-specific targeting moiety
to produce a tri-fusion molecule. This molecule can bind to a leukocyte
surface molecule, passes through the membrane and targets PDZ domains.
Alternatively, a PDZ motif sequence (PL sequence) may be linked to a
cell-specific targeting moiety that binds to a surface molecule that
internalizes the fusion peptide.

[0455]In an other approach, microspheres of artificial polymers of mixed
amino acids (proteinoids) have been used to deliver pharmaceuticals. For
example, U.S. Pat. No. 4,925,673 describes drug-containing proteinoid
microsphere carriers as well as methods for their preparation and use.
These proteinoid microspheres are useful for the delivery of a number of
active agents. Also see, U.S. Pat. Nos. 5,907,030 and 6,033,884, which
are incorporated herein by reference.

[0456]6.9.2 Introduction of Polynucleotides into Cells

[0457]A polynucleotide encoding a surface receptor C-terminal peptide may
be useful in the treatment of various leukocyte activation-associated
abnormal conditions. By introducing gene sequences into cells, gene
therapy can be used to treat conditions in which leukocytes are activated
to result in deleterious consequences. In one embodiment, a
polynucleotide that encodes a PL sequence peptide of the invention is
introduced into a cell where it is expressed. The expressed peptide then
inhibits the interaction of PDZ proteins and PL proteins in the cell.

[0458]Thus, in one embodiment, the polypeptides of the invention are
expressed in a cell by introducing a nucleic acid (e.g., a DNA expression
vector or mRNA) encoding the desired protein or peptide into the cell.
Expression may be either constitutive or inducible depending on the
vector and choice of promoter. Methods for introduction and expression of
nucleic acids into a cell are well known in the art and described herein.

[0459]In a specific embodiment, nucleic acids comprising a sequence
encoding a peptide disclosed herein, are administered to a human subject.
In this embodiment of the invention, the nucleic acid produces its
encoded product that mediates a therapeutic effect by inhibiting
leukocyte activation. Any of the methods for gene therapy available in
the art can be used according to the present invention. Exemplary methods
are described below.

[0461]In a preferred embodiment of the invention, the therapeutic
composition comprises a coding sequence that is part of an expression
vector. In particular, such a nucleic acid has a promoter operably linked
to the coding sequence, said promoter being inducible or constitutive,
and, optionally, tissue-specific. In another specific embodiment, a
nucleic acid molecule is used in which the coding sequence and any other
desired sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the nucleic acid (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,
Nature 342:435-438).

[0462]Delivery of the nucleic acid into a patient may be either direct, in
which case the patient is directly exposed to the nucleic acid or nucleic
acid-carrying vector, or indirect, in which case, cells are first
transformed with the nucleic acid in vitro, then transplanted into the
patient. These two approaches are known, respectively, as in vivo or ex
vivo gene therapy.

[0463]In a specific embodiment, the nucleic acid is directly administered
in vivo, where it is expressed to produce the encoded product. This can
be accomplished by any methods known in the art, e.g., by constructing it
as part of an appropriate nucleic acid expression vector and
administering it so that it becomes intracellular, e.g., by infection
using a defective or attenuated retroviral or other viral vector (see
U.S. Pat. No. 4,980,286), by direct injection of naked DNA, by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), by
coating with lipids or cell-surface receptors or transfecting agents, by
encapsulation in liposomes, microparticles, or microcapsules, by
administering it in linkage to a peptide which is known to enter the
nucleus, or by administering it in linkage to a ligand subject to
receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem.
262:4429-4432) which can be used to target cell types specifically
expressing the receptors. In another embodiment, a nucleic acid-ligand
complex can be formed in which the ligand comprises a fusogenic viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid
lysosomal degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by targeting a
specific receptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16,
1992; WO 92/22635 dated Dec. 23, 1992; WO92/20316 dated Nov. 26, 1992;
WO93/14188 dated Jul. 22, 1993; WO 93/20221 dated Oct. 14, 1993).
Alternatively, the nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, byhomologous
recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0464]In a preferred embodiment of the invention, adenoviruses as viral
vectors can be used in gene therapy. Adenoviruses have the advantage of
being capable of infecting non-dividing cells (Kozarsky and Wilson, 1993,
Current Opinion in Genetics and Development 3:499-503). Other instances
of the use of adenoviruses in gene therapy can be found in Rosenfeld et
al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234. Furthermore,
adenoviral vectors with modified tropism may be used for cell specific
targeting (WO98/40508). Adeno-associated virus (AAV) has also been
proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp.
Biol. Med. 204:289-300).

[0465]In addition, retroviral vectors (see Miller et al., 1993, Meth.
Enzymol. 217:581-599) have been modified to delete retroviral sequences
that are not necessary for packaging of the viral genome and integration
into host cell DNA. The coding sequence to be used in gene therapy is
cloned into the vector, which facilitates delivery of the gene into a
patient. More detail about retroviral vectors can be found in Boesen et
al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral
vector to deliver the mdr1 gene to hematopoietic stem cells in order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the use of retroviral vectors in gene therapy are: Clowes et
al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood
83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141;
and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.

[0466]Another approach to gene therapy involves transferring a gene to
cells in tissue culture. Usually, the method of transfer includes the
transfer of a selectable marker to the cells. The cells are then placed
under selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to a
patient.

[0467]In this embodiment, the nucleic acid is introduced into a cell prior
to administration in vivo of the resulting recombinant cell. Such
introduction can be carried out by any method known in the art, including
but not limited to transfection, electroporation, lipofection,
microinjection, infection with a viral or bacteriophage vector containing
the nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
Numerous techniques are known in the art for the introduction of foreign
genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol.
217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985,
Pharmac. Ther. 29:69-92) and may be used in accordance with the present
invention, provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique should
provide for the stable transfer of the nucleic acid to the cell, so that
the nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell progeny. In a preferred embodiment, the cell used
for gene therapy is autologous to the patient.

[0468]In a specific embodiment, the nucleic acid to be introduced for
purposes of gene therapy comprises an inducible promoter operably linked
to the coding sequence, such that expression of the nucleic acid is
controllable by controlling the presence or absence of the appropriate
inducer of transcription.

[0469]Oligonucleotides such as anti-sense RNA and DNA molecules, and
ribozymes that function to inhibit the translation of a leukocyte surface
receptor mRNA, especially its C-terminus are also within the scope of the
invention. Anti-sense RNA and DNA molecules act to directly block the
translation of mRNA by binding to targeted mRNA and preventing protein
translation. In regard to antisense DNA, oligodeoxyribonucleotides
derived from the translation initiation site, e.g., between -10 and +10
regions of a nucleotide sequence, are preferred.

[0471]Ribozymes are enzymatic RNA molecules capable of catalyzing the
specific cleavage of RNA. The mechanism of ribozyme action involves
sequence specific hybridization of the ribozyme molecule to complementary
target RNA, followed by endonucleolytic cleavage. Within the scope of the
invention are engineered hammerhead motif ribozyme molecules that
specifically and efficiently catalyze endonucleolytic cleavage of
leukocyte surface receptor RNA sequences.

[0472]Specific ribozyme cleavage sites within any potential RNA target are
initially identified by scanning the target molecule for ribozyme
cleavage sites which include the following sequences, GUA, GUU and GUC.
Once identified, short RNA sequences of between 15 and 20 ribonucleotides
corresponding to the region of the target gene containing the cleavage
site may be evaluated for predicted structural features such as secondary
structure that may render the oligonucleotide sequence unsuitable. The
suitability of candidate targets may also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides, using
ribonuclease protection assays.

[0473]The anti-sense RNA and DNA molecules and ribozymes of the invention
may be prepared by any method known in the art for the synthesis of
nucleic acid molecules. These include techniques for chemically
synthesizing oligodeoxyribonucleotides well known in the art such as for
example solid phase phosphoramidite chemical synthesis. Alternatively,
RNA molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the RNA molecule. Such DNA sequences may be
incorporated into a wide variety of vectors which contain suitable RNA
polymerase promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense RNA
constitutively or inducibly, depending on the promoter used, can be
introduced stably into cell lines.

[0474]Various modifications to the DNA molecules may be introduced as a
means of increasing intracellular stability and half-life. Possible
modifications include, but are not limited to, the addition of flanking
sequences of ribo- or deoxy-nucleotides to the 5' and/or 3' ends of the
molecule or the use of phosphorothioate or 2'O-methyl rather than
phospho-diesterase linkages within the oligodeoxyribonucleotide backbone.

[0475]6.9.3 Other Pharmaceutical Compositions

[0476]The compounds of the invention, may be administered to a subject per
se or in the form of a sterile composition or a pharmaceutical
composition. Pharmaceutical compositions comprising the compounds of the
invention may be manufactured by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes. Pharmaceutical
compositions may be formulated in conventional manner using one or more
physiologically acceptable carriers, diluents, excipients or auxiliaries
that facilitate processing of the active peptides or peptide analogues
into preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.

[0477]For topical administration the compounds of the invention may be
formulated as solutions, gels, ointments, creams, suspensions, etc. as
are well-known in the art.

[0478]Systemic formulations include those designed for administration by
injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or
intraperitoneal injection, as well as those designed for transdermal,
transmucosal, oral or pulmonary administration.

[0479]For injection, the compounds of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers such
as Hanks's solution, Ringer's solution, or physiological saline buffer.
The solution may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents.

[0480]Alternatively, the compounds may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0481]For transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such penetrants are
generally known in the art. This route of administration may be used to
deliver the compounds to the nasal cavity.

[0482]For oral administration, the compounds can be readily formulated by
combining the active peptides or peptide analogues with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the like, for
oral ingestion by a patient to be treated. For oral solid formulations
such as, for example, powders, capsules and tablets, suitable excipients
include fillers such as sugars, such as lactose, sucrose, mannitol and
sorbitol; cellulose preparations such as maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If
desired, disintegrating agents may be added, such as the cross-linked
polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as
sodium alginate.

[0483]If desired, solid dosage forms may be sugar-coated or enteric-coated
using standard techniques.

[0485]For buccal administration, the compounds may take the form of
tablets, lozenges, etc. formulated in conventional manner.

[0486]For administration by inhalation, the compounds for use according to
the present invention are conveniently delivered in the form of an
aerosol spray from pressurized packs or a nebulizer, with the use of a
suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol the dosage unit
may be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of e.g. gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the compound and
a suitable powder base such as lactose or starch.

[0487]The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other glycerides.

[0488]In addition to the formulations described previously, the compounds
may also be formulated as a depot preparation. Such long acting
formulations may be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection. Thus,
for example, the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable oil)
or ion exchange resins, or as sparingly soluble derivatives, for example,
as a sparingly soluble salt.

[0489]Alternatively, other pharmaceutical delivery systems may be
employed. Liposomes and emulsions are well known examples of delivery
vehicles that may be used to deliver peptides and peptide analogues of
the invention. Certain organic solvents such as dimethylsulfoxide also
may be employed, although usually at the cost of greater toxicity.
Additionally, the compounds may be delivered using a sustained-release
system, such as semipermeable matrices of solid polymers containing the
therapeutic agent. Various of sustained-release materials have been
established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days. Depending on
the chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be employed.

[0490]As the compounds of the invention may contain charged side chains or
termini, they may be included in any of the above-described formulations
as the free acids or bases or as pharmaceutically acceptable salts.
Pharmaceutically acceptable salts are those salts which substantially
retain the biologic activity of the free bases and which are prepared by
reaction with inorganic acids. Pharmaceutical salts tend to be more
soluble in aqueous and other protic solvents than are the corresponding
free base forms.

6.10. EFFECTIVE DOSAGES

[0491]The compounds of the invention will generally be used in an amount
effective to achieve the intended purpose. For use to inhibit leukocyte
activation-associated disorders, the compounds of the invention or
pharmaceutical compositions thereof, are administered or applied in a
therapeutically effective amount. By therapeutically effective amount is
meant an amount effective ameliorate or prevent the symptoms, or prolong
the survival of, the patient being treated. Determination of a
therapeutically effective amount is well within the capabilities of those
skilled in the art, especially in light of the detailed disclosure
provided herein. An "inhibitory amount" or "inhibitory concentration" of
a PL-PDZ binding inhibitor is an amount that reduces binding by at least
about 40%, preferably at least about 50%, often at least about 70%, and
even as much as at least about 90%. Binding can as measured in vitro
(e.g., in an A assay or G assay) or in situ.

[0492]For systemic administration, a therapeutically effective dose can be
estimated initially from in vitro assays. For example, a dose can be
formulated in animal models to achieve a circulating concentration range
that includes the IC50 as determined in cell culture (i.e., the
concentration of test compound that inhibits 50% of leukocyte surface
receptor-PDZ domain-containing protein interactions). Such information
can be used to more accurately determine useful doses in humans.

[0493]Initial dosages can also be estimated from in vivo data, e.g.,
animal models, using techniques that are well known in the art. One
having ordinary skill in the art could readily optimize administration to
humans based on animal data.

[0494]Dosage amount and interval may be adjusted individually to provide
plasma levels of the compounds that are sufficient to maintain
therapeutic effect. Usual patient dosages for administration by injection
range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1
mg/kg/day. Therapeutically effective serum levels may be achieved by
administering multiple doses each day.

[0495]In cases of local administration or selective uptake, the effective
local concentration of the compounds may not be related to plasma
concentration. One having skill in the art will be able to optimize
therapeutically effective local dosages without undue experimentation.

[0496]The amount of compound administered will, of course, be dependent on
the subject being treated, on the subject's weight, the severity of the
affliction, the manner of administration and the judgment of the
prescribing physician.

[0497]The therapy may be repeated intermittently while symptoms detectable
or even when they are not detectable. The therapy may be provided alone
or in combination with other drugs. In the case of conditions associated
with leukocyte activation such as transplantation rejection and
autoimmunity, the drugs that may be used in combination with the
compounds of the invention include, but are not limited to, steroid and
non-steroid anti-inflammatory agents.

[0498]6.10.1 Toxicity

[0499]Preferably, a therapeutically effective dose of the compounds
described herein will provide therapeutic benefit without causing
substantial toxicity.

[0500]Toxicity of the compounds described herein can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., by determining the LD50 (the dose lethal to 50% of
the population) or the LD100 (the dose lethal to 100% of the
population). The dose ratio between toxic and therapeutic effect is the
therapeutic index. Compounds which exhibit high therapeutic indices are
preferred. The data obtained from these cell culture assays and animal
studies can be used in formulating a dosage range that is not toxic for
use in human. The dosage of the compounds described herein lies
preferably within a range of circulating concentrations that include the
effective dose with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration
and dosage can be chosen by the individual physician in view of the
patient's condition. (See, e.g., Fingl et al., 1975, In: The
Pharmacological Basis of Therapeutics, Ch. 1, p. 1).

[0504]Supernatants were assayed for cytokine production following
activation of T cell lines. Mouse T cell lines were stimulated using two
different methods, either with antigen and antigen presenting cells or
anti-mouse CD3.

[0506]Antibody against mouse CD3 (Pharmigen #145-2C11) was coated
overnight at 4° C. using 96-well flat bottom Elisa plates at a
final concentration of 0.5 μg/ml, diluted in PBS. Just prior to use,
plates were washed three times with 200 μl/well PBS to remove excess
anti-CD3. To ensure that cells were given sufficient time to transduce
Tat-peptides before activation, T cells (5×105 cells/ml) were
pre-treated with or without 10 μM Tat-peptides for two hours at 5%
CO2, 37° C. and then diluted in media with or without 10 μM
Tat-peptides to a final concentration of 2×104 cells per well
in a final volume of 200 μl. Cells were then treated as described
above.

6.11.1.4 Cytokine ELISA

[0507]IFNγ was measured from cell supernatants, described above, at
ambient temperature using the Endogen, Inc. ELISA protocol 3. Briefly,
96-well, flat bottom, high binding ELISA plates were preincubated
overnight with coating antibody (MM700). Plates were washed with 50 mM
TRIS, 0.2% tween-20, pH 8 and they blocked for one hour with PBS plus 2%
BSA. Washed plates were then incubated one hour with 25 μl of cell
supernatant and 25 μl blocking buffer, or with 50 μl IFNγ
standard. The presence of IFNγ was detected with a biotin-labeled
anti-mouse IFNγ monoclonal antibody (MM700B, Endogen, Inc.).
Quantitative amounts of detection antibody are revealed with horseradish
peroxidase-conjugated streptavidin. The enzymatic, color, substrate for
HRP, tetramethylbenzidine (TMB), was developed for up to 30 minutes and
stopped with 1.0 M H2SO4. The absorbance at 450 nm was measured
using a microtiter plate reader (Thermo Max, Molecular Devices) and the
concentration of unknown IFNγ from cell supernatants was calculated
from a standard curve generated by Softmax Pro. software (Molecular
Devices).

Design of an Inhibitor of DLG 1-Ligand Binding with Greater than 100 μM
Potency

[0510]A GST/DLG1 fusion protein (See TABLE 3) and a biotin-labeled peptide
corresponding to the C-terminal 20 amino acids of the CLASP-2 protein,
peptide AA2L (See TABLE 4), were synthesized and purified by standard
techniques well known in the art as described supra. This PDZ-ligand
combination was then shown to bind specifically using both the "A" assay
and the "G" assay (See TABLE 2). Once specific binding was demonstrated,
the apparent affinity of the binding interaction was determined using
Approach 1 of the section entitled "Measurement of PDZ-ligand binding
affinity" (see FIG. 2A). The measured apparent affinity was 21 μM.
This implies that 21 μM labeled CLASP-2 peptide AA2L filled 50% of the
binding sites for CLASP-2 on DLG1. Thus, 21 μM unlabeled CLASP-2
peptide should be able to block the binding of a given ligand to DLG1 by
approximately 50%, assuming that the given ligand (1) binds to the same
site(s) on DLG1 as Qasp˜2 and (2) is not added at sufficient
concentration to reduce significantly the binding of the CLASP-2 peptide
(i.e. cannot out-compete the CLASP-2 peptide).

[0511]To detect such inhibition, it was necessary to synthesize an
analogue of the CLASP2peptide AA2L that (1) retained similar DLG1 binding
properties and (2) would not itself generate a signal in the assay
selected to measure inhibition. Because most molecular interactions
between PDZ proteins and their ligands involve only the C-terminal 6
amino acids of the ligand, an eight amino acid variant of the CLASP-2
peptide, MTSSSSVV (SEQ ID NO:227), was anticipated to retain similar DLG1
binding properties as the 20 amino acid AA2L CLASP-2 peptide. This eight
amino acid CLASP-2 peptide (lacking a functional label) was therefore
synthesized and purified by standard techniques as described supra. When
100 μM of the (functionally unlabeled) eight amino acid CLASP-2
peptide and 20 μM of the biotin-labeled AA2L CLASP-2 peptide were
added simultaneously to DLG1 in a variant of the "G" assay (described
supra), the binding of the labeled AA2L CLASP-2 peptide was, as
predicted, inhibited by greater than 50% (FIG. 3A). An analogous
experiment in which the labeled AA2L CLASP-2 peptide was replaced with
another labeled DLG1 ligand, labeled AAI3L Fas peptide demonstrated
similar inhibition by the eight amino acid CLASP-2 peptide (FIG. 3A).
Thus, an effective inhibitor of DLG1-ligand binding (i.e. the eight amino
acid CLASP-2 peptide MTSSSSVV (SEQ ID NO:227)) with a known potency range
(order of magnitude 21 μM) was designed based on knowledge of the
affinity, 21 μM, with which a particular labeled ligand, the CLASP-2
peptide AA2L, bound to DLG1.

[0512]The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of single
aspects of the invention and any sequences which are functionally
equivalent are within the scope of the invention. Indeed, various
modifications of the invention in addition to those shown and described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.

[0513]All publications cited herein are incorporated by reference in their
entirety and for all purposes.